Method of treating an ischemia-reperfusion injury-related disorder by administering GPCR ligands

ABSTRACT

Disclosed are peptide ligands for G-protein coupled receptors that are useful for treating disorders associated with G-protein coupled receptor activation.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/901,975 filed Sep. 18, 2007, now U.S. Pat. No. 7,884,180, whichclaims the benefit of priority of U.S. Ser. No. 60/845,606, filed Sep.18, 2006; U.S. Ser. No. 60/846,421, filed Sep. 21, 2006; and U.S. Ser.No. 60/851,591, filed Oct. 12, 2006. The contents of all of theseapplications are incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The invention relates to bioactive peptides.

BACKGROUND OF THE INVENTION

Known and uncharacterized GPCRs (G-protein coupled receptors) currentlyconstitute major targets for drug action and development, and >30% ofall marketed therapeutics act on them (Jacoby et al 2006, ChemMedChem 1,760-782). GPCRs usually have seven transmembrane domains. Upon bindingof a ligand to an extra-cellular portion or fragment of a GPCR, a signalis transduced within the cell that results in a change in a biologicalor physiological property or behavior of the cell. GPCRs, along withG-proteins and effectors (intracellular enzymes and channels modulatedby G-proteins), are the components of a modular signaling system thatconnects the state of intra-cellular second messengers to extra-cellularinputs (Pierce et al 2002, Nature Reviews Molecular Cell Biology 3,639-650). The GPCRs seem to be of critical importance to both thecentral nervous system and peripheral physiological processes.

The GPCR superfamily is diverse and sequencing of the human genome hasrevealed >850 genes that encode them (Hopkins and Groom 2002, NatureReviews Drug Discovery 1, 727-730). There is great diversity within theGPCRs, which is matched by a great variety of ligands that activatethem. Known drugs target only ˜30 members of the GPCR family, mainlybiogenic amine receptors. Thus, there is an enormous potential withinthe pharmaceutical industry to exploit the remaining family members,including the >100 orphan receptors for which no existing ligands haveso far been identified (Gilchrist 2004, Expert Opin. Ther. Targets 8,495-498).

There are ongoing efforts to identify new GPCRs and to deorphanize knownGPCRs, which can be used to screen for new agonists and antagonistshaving potential prophylactic and therapeutical properties (Gilchrist2004, Expert Opin. Ther. Targets 8, 495-498; Schyler and Horuk 2006,Drug Discovery Today 11, 481-493). Below are examples of GPCRs pertinentto this application, which may serve as targets for novel therapeuticagents.

The Mas receptor is the product of the MAS1 proto-oncogene, which wasfirst isolated based on its tumorigenic activity and later identified asa member of the rhodopsin-like class A GPCR subfamily. It was recentlydemonstrated that Ang(1-7) is an agonist of Mas and that this peptide isformed by the action of ACE2 (angiotensin converting enzyme 2) onangiotensin I (reviewed in Santos et al. 2007, Current CardiologyReviews 3, 57-64). While the chronic increase in AngII can induce manydeleterious effects on the heart, Ang(1-7) has cardioprotective actions,including vasodilation and antiproliferative activities, which oftenoppose those of AngII. The effects of Ang(1-7) are associated withlowering blood pressure, prevention of cardiac remodeling andattenuation of renal abnormalities associated with hypertension(Reudelhuber 2006, Hypertension 47, 811-815).

Mas, ACE2 and Ang(1-7) are considered important components of therenin-angiotensin system (RAS), which is a major regulator ofcardiovascular homeostasis and hydroelectrolyte balance (Silva et al2006, Mini-reviews in Medicinal Chemistry 6, 603-609; Santos andFerreira 2007, Current Opinion in Nephrology and Hypertension 16,122-128). Disturbances in the RAS system play a pivotal role in thepathogenesis of hypertension and cardiovascular diseases. RAS can beviewed as a system comprising two main axes with opposite actions: thevasoconstrictor/proliferative ACE-AngII-AT1/AT2 axis, and thevasodilator/anti-proliferative ACE2-Ang(1-7)-Mas axis.

Components of the ACE-AngII-AT1/AT2 axis serve as targets for two majortypes of drugs, the ACE inhibitors (ACEi) and the AT1 receptor blockers(ARBs), which are successful therapeutic strategies against severalclinical conditions, including arterial hypertension, left ventricularsystolic dysfunction, chronic heart failure, myocardial infarction anddiabetic and non-diabetic chronic kidney diseases (Ferrario 2006,Journal of the Renin-Angiotensin-Aldosterone System 7, 3-14). TheACE2-Ang(1-7)-Mas axis is considered as a putatively important targetfor the development of new drugs to treat cardiovascular and renaldiseases (Santos et al. 2007, Current Cardiology Reviews 3, 57-64;Keidar et al 2007, Cardiovascular Research 73, 463-469). The potentialtherapeutic application of the Mas receptor is indeed supported by thecardioprotective and beneficial effects of its peptide agonist,Ang(1-7), and an orally active nonpeptide agonist, AVE 0991, in severalexperimental models (Santos and Ferreira 2006, Cardiovascular DrugReviews 24, 239-246).

The FPRL1 receptor belongs to the FPR (formyl-peptide receptor) relatedfamily of GPCRs that also includes FPR and FPRL2 (Le et al 2001,Cytokine and Growth Factor Reviews 12, 91-105). This receptor, alsoknown as the lipoxin A₄ receptor, ALXR, binds pleiotropic ligands, i.e.both lipids and peptides, and is expressed primarily by neutrophils,eosinophils and monocytes (Chiang et al 2006, Pharmacological Reviews58, 463-487). The two prominent types of endogenous FPRL1 ligands,lipoxin A₄ (LXA4) and the aspirin-triggered lipoxins (ATLs), and theAnnexinI protein and its N-terminal derived peptides, have shownanti-inflammatory properties in various experimental animal models(Gavins et al 2005, Prostaglandins, Leukotrienes and Essential FattyAcids 73, 211-219).

Extensive research has clarified that the mechanism underlying theanti-inflammatory activity gained upon FPRL1 activation by these ligandsis achieved by promoting resolution of inflammation—an active andtightly synchronized process, involving counter-regulation of leukocytes(Scannell and Maderna 2006, The Scientific World Journal 6, 1555-1573).Activation of FPRL1 evokes inhibition of polymorphonuclear neutrophils(PMN's) and eosinophils migration and prevents leukocyte-mediated tissueinjury. In addition, emigration of monocytes is stimulated upon FPRL1activation, enabling the clearance of apoptotic cells from theinflammatory site in a nonphlogistic manner. Furthermore, NKcytotoxicity is inhibited which further contributes to downregulation ofproinflammatory mediators at the site of inflammation.

Both LXA4 (and its stable analog ATLa) and Ac2-26 (a peptide derivedfrom the N-terminus of AnnexinI) have been extensively studied invarious animal disease models of acute and chronic inflammation, such asdermal inflammation, colitis, asthma, and ischemia/reperfusion injury,and were found to be efficacious (Perretti and Gavins 2003, NewsPhysiol. Sci. 18, 60-64; Gewirtz 2005, Current Opinion inInvestigational Drugs 6, 1112-1115). These findings indicate that FPRL1agonists open new avenues and approaches to therapeutic interventionsvia accelerated resolution of inflammation, and might have a beneficialtherapeutic value in various pathological inflammatory conditions, suchas ischemia/reperfusion injury, organ transplantation, inflammatorybowel disease, psoriasis, asthma, and arthritis. A stable lipoxin analogis indeed in clinical development for inflammatory bowel disease(Berlex).

The MrgX1 (Mas-related gene X1) receptor, also named SNSR4 (sensoryneuron specific receptor 4), has been detected only in the nociceptivesensory neurons of the dorsal root ganglia (Dong et al 2001, Cell 106,619-632). It is preferentially activated by opioid-related peptides,such as the proenkephalin A-derived peptide, BAM22 (Lembo et al 2002,Nature Neurosci. 5, 201-209). MrgX2 is another member of the Mrg familyof GPCRs, which also shows high expression in nociceptive neurons, butalso in various other tissues. Various high and low affinity ligandshave been identified for MrgX2 (Robas et al 2003, J. Biol. Chem. 278,44400-44404). The physiological role that these receptors play in vivois not clear.

Based on their expression in nociceptors, which are neurons that mediatenociceptive transmission of pain, Mrg receptors are believed to play arole in the sensation or modulation of acute pain as well as chronicpain associated with nerve injury or inflammation. The Mrg family, andin particular MrgX1, are thus viewed as promising pharmacologicaltargets for the management of pain (Ahmad and Dray 2004; Current Opinionin Investigational Drugs 5, 67-70; Dray 2003, Current Opinion inAnesthesiology 16, 521-525).

MrgX2 is also activated by several secretagogues, and seems toparticipate in the activation of human mast cells by such substancesTatemoto et al 2006, Biochem. Biophys. Res. Comm. 349, 1322-1328). Assuch, MrgX2 might provide a novel therapeutic target for the control ofdiseases involving mast cell activation. CST, a high affinity ligand ofMrgX2, is a neuropeptide involved in sleep regulation and locomotoractivity (Robas et al 2003, J. Biol. Chem. 278, 44400-44404). CST alsoemerged as a potential endogenous immune modulator, and has recentlyshown potent anti-inflammatory activity in experimental animal models(Gonzalez-Rey and Delgado 2006, Drug News Perspect 19, 393-399). MrgX2may thus also be involved in sleep regulation, and in inflammation.

Another high affinity ligand of MrgX2 is PAMP-12 (Kamohara et al 2005,Biochem. Biophys. Res. Comm. 330, 1146-1152; Nothacker et al 2005, Eur.J. Pharmacol. 519, 191-193), which derives from proadrenomedullin andlike other PAMP peptides has vasophysiological functions that appear torelate to several diseases, such as hypertension, chronic renal failureand congestive heart failure and chronic glomerulonephritis (Kobayashiet al 2003, Hypertension Research 26, S71-S78). Based on this, MrgX2 mayalso be a target of potential hypotension-regulating drugs.

SUMMARY OF THE INVENTION

The invention is based in part on the identification of novel peptides:Peptide 33-type peptides, Peptide 58-type peptides, Peptide 60-typepeptides, Peptide 61-type peptides, Peptide 63-type peptides, andPeptide 94-type peptides, that act as ligands of four known GPCRreceptors, but do not show significant homology to known GPCR ligands.

Two peptides, exemplified by Peptide 61_S and Peptide 33_V and compoundsof Formulas I and II, respectively, activate the MAS1 gene product, i.e.the Mas receptor. This receptor is an important component of the reninangiotensin system, which is a major regulator of cardiovascularhomeostasis and hydroelectrolyte balance. This receptor is viewed as aputatively important target for the development of new drugs to treatcardiovascular and renal diseases. As is explained below, peptidesrepresented by Peptide 61_S and 33_V act as agonists of the Masreceptor, and elicit calcium flux in Mas-expressing cells. Furthermore,these peptides induce relaxation of rat aortic rings and reduced hearthypertrophy induced by Isoproterenol. Thus, peptides represented byPeptide 61_S and 33_V, as well as compounds within Formulas I and II,are useful as agonists in conditions benefiting from increasing theactivity of the Mas receptor, such as hypertension, heart failure andother cardiovascular pathological conditions.

The peptide exemplified by Peptide 60_S falls within a compound ofFormula III and activates the MRGPRX1 gene product, i.e the MrgX1receptor (Mas-related G-protein coupled receptor member X1, also knownas SNSR4). This receptor is believed to be involved in the function ofnociceptive neurons and to regulate nociceptor function and/ordevelopment, including the sensation or modulation of pain. It ispotently activated by enkephalins-derived peptides, such as BAM22(bovine adrenal medulla peptide 22). Peptide 60_S is superior to BAM22in eliciting calcium flux in MrgX1 expressing cells. Thus, as isexplained below, peptides represented by Peptide 60_S, as well ascompounds within Formula III, are useful as agonists in conditionsbenefiting from increasing the activity of MrgX1.

Peptides exemplified by Peptide 61_S, Peptide 60_S, Peptide 94, andPeptide 63, and compounds of Formulas I, III, IV, and V, respectively,activate the MRGPRX2 gene product, MrgX2 (Mas-related G-protein coupledreceptor member X2). This receptor is believed to be involved in thefunction of nociceptive neurons and to regulate nociceptor functionand/or development, including the sensation or modulation of pain.Cortistatin-14 (CST) is a high potency ligand of this receptor.Cortistatin has several biological functions, including roles in sleepregulation, locomotor activity, and cortical function. Peptide 60_S,Peptide 61_S, Peptide 63, and Peptide 94 are superior to cortistatin-14in eliciting calcium flux in MrgX2-expressing cells. Thus, as explainedbelow, peptides represented by Peptide 61_S, Peptide 60_S, Peptide 94,and Peptide 63, as well as compounds within Formulas I, III, IV, and V,respectively, are useful as agonists in conditions benefiting fromincreasing the activity of MrgX2.

Peptides exemplified by Peptide 33_V, Peptide 60_S, Peptide 94, andPeptide 58, and compounds of Formulas II, III, IV, and VI, respectively,activate FPRL1 (FPR-related receptor 1) (also known as lipoxin A₄receptor, LXA4R or ALXR). The activation of FPRL1 by lipoxins orannexinI-derived peptides results in anti-inflammatory effects. Peptide33_V, Peptide 60_S, Peptide 94, and Peptide 58 induce calcium flux inFPRL-1 expressing cells. Furthermore, Peptide 58 and peptides derivedfrom it, exhibit anti-inflammatory activity in a mouse model of acuteinflammation. Thus, as explained below, peptides represented by Peptide33_V, Peptide 60_S, Peptide 94, and Peptide 58, as well as compoundswithin Formulas II, III, IV, and VI, are useful as agonists inconditions benefiting from increasing the activity of a FPRL1, such asacute and chronic inflammation.

In addition the invention includes the following additional embodiments.

The invention in one embodiment includes a peptide less than 100 aminoacids in length, said polypeptide comprising the amino acid sequence ofFormula I, Formula II, Formula III, Formula IV, Formula V, or FormulaVI, wherein Formula I is

-   A¹-A²-A³-A⁴-A⁵-A⁶-A⁷-A⁸-A⁹-A¹⁰-A¹¹-A^(l2)-A¹³-A¹⁴-A¹⁵-A¹⁶-A¹⁷-A¹⁸-A¹⁹-A²⁰-A²¹-A²²-A²³-A²⁴-A²⁵-A²⁶,    or a pharmaceutically acceptable salt thereof; wherein    -   A¹ is absent or F or a hydrophobic non-naturally occurring amino        acid;    -   A² is absent or A or a small non-naturally occurring amino acid;    -   A³ is absent or F or a hydrophobic non-naturally occurring amino        acid;    -   A⁴ is absent or L or a hydrophobic non-naturally occurring amino        acid;    -   A⁵ is absent or G or a small non-naturally occurring amino acid;    -   A⁶ is absent or Y or a hydrophobic non-naturally occurring amino        acid;    -   A⁷ is absent or S or C;    -   A⁸ is absent or I or a hydrophobic non-naturally occurring amino        acid;    -   A⁹ is absent or Y or a hydrophobic non-naturally occurring amino        acid;    -   A¹⁰ is absent or L or a hydrophobic non-naturally occurring        amino acid;    -   A¹¹ is absent or N or a polar non-naturally occurring amino        acid;    -   A^(l2) is absent or R or a basic non-naturally occurring amino        acid;    -   A¹³ is absent or K or a basic non-naturally occurring amino        acid;    -   A¹⁴ is absent or R or a basic non-naturally occurring amino        acid;    -   A¹⁵ is absent or R or a basic non-naturally occurring amino        acid;    -   A¹⁶ is absent or G, or a small non-naturally occurring amino        acid;    -   A¹⁷ is absent or D or a polar non-naturally occurring amino        acid;    -   A¹⁸ is absent or P;    -   A¹⁹ is absent or A or a hydrophobic non-naturally occurring        amino acid;    -   A²⁰ is absent or F or a hydrophobic non-naturally occurring        amino acid;    -   A²¹ is absent or K or a basic non-naturally occurring amino        acid;    -   A²² is absent or R or a basic non-naturally occurring amino        acid;    -   A²³ is absent or R or a basic non-naturally occurring amino        acid;    -   A²⁴ is absent or L or a hydrophobic non-naturally occurring        amino acid;    -   A²⁵ is absent or R or a hydrophobic non-naturally occurring        amino acid;    -   A²⁶ is absent or D or a polar non-naturally occurring amino        acid;        wherein Formula II is-   B¹-B²-B³-B⁴-B⁵-B⁶-B⁷-B⁸-B⁹-B¹⁰-B¹¹-B¹²-B¹³-B¹⁴-B¹⁵-B¹⁶-B¹⁷-B¹⁸, or a    pharmaceutically acceptable salt thereof;

wherein

-   -   B¹ is absent or S;    -   B² is absent or M or norleucine (Nle) or another hydrophobic        non-naturally occurring amino acid;    -   B³ is absent or C or V;    -   B⁴ is absent or H or a basic non-naturally occurring amino acid;    -   B⁵ is absent or R or a basic non-naturally occurring amino acid;    -   B⁶ is absent or W or a hydrophobic non-naturally occurring amino        acid    -   B⁷ is absent or S;    -   B⁸ is absent or R or a hydrophobic non-naturally occurring amino        acid;    -   B⁹ is A or a small non-naturally occurring amino acid;    -   B¹⁰ is V or a hydrophobic non-naturally occurring amino acid;    -   B¹¹ is L or a hydrophobic non-naturally occurring amino acid;    -   B¹² is F or a hydrophobic non-naturally occurring amino acid;    -   B¹³ is P;    -   B¹⁴ is A or a hydrophobic non-naturally occurring amino acid;    -   B¹⁵ is A or a hydrophobic non-naturally occurring amino acid;    -   B¹⁶ is H or a basic non-naturally occurring amino acid;    -   B¹⁷ is R or a basic non-naturally occurring amino acid;    -   B¹⁸ is P;        wherein Formula III is

-   C¹-C²-C³-C⁴-C⁵-C⁶-C⁷-C⁸-C⁹-C¹⁰-C¹¹-C¹²-C¹³-C¹⁴-C¹⁵-C¹⁶-C¹⁷-C¹⁸-C¹⁹-C²⁰-C²¹-C²²-C²³-C²⁴-C²⁵-C²⁶-C²⁷-C²⁸,    or a pharmaceutically acceptable salt thereof;    -   C¹ is absent or G or a small non-naturally occurring amino acid;    -   C² is absent or I or a hydrophobic non-naturally occurring amino        acid;    -   C³ is absent or G or a small non-naturally occurring amino acid;        ;    -   C⁴ is C or S or a polar non-naturally occurring amino acid;    -   C⁵ is V or a hydrophobic non-naturally occurring amino acid;    -   C⁶ is W or a hydrophobic non-naturally occurring amino acid    -   C⁷ is H or a basic non-naturally occurring amino acid;    -   C⁸ is W or a hydrophobic non-naturally occurring amino acid;    -   C⁹ is K or a basic non-naturally occurring amino acid;    -   C¹⁰ is H or a basic non-naturally occurring amino acid;    -   C¹¹ is R or a basic non-naturally occurring amino acid;    -   C¹² is V or a hydrophobic non-naturally occurring amino acid;    -   C¹³ is A or a hydrophobic non-naturally occurring amino acid;    -   C¹⁴ is T or a polar non-naturally occurring amino acid;    -   C¹⁵ is R or a basic non-naturally occurring amino acid;    -   C¹⁶ is F or a hydrophobic non-naturally occurring amino acid;    -   C¹⁷ is T or a polar non-naturally occurring amino acid;    -   C¹⁸ is L or a hydrophobic non-naturally occurring amino acid;    -   C¹⁹ is P;    -   C²⁰ is R or basic non-naturally occurring amino acid;    -   C²¹ is F or a polar non-naturally occurring amino acid;    -   C²² is L or a hydrophobic non-naturally occurring amino acid;    -   C²³ is Q or a polar non-naturally occurring amino acid.    -   C²⁴ is absent or R or a basic non-naturally occurring amino        acid;    -   C²⁵ is absent or R or a basic non-naturally occurring amino        acid;    -   C²⁶ is absent or S or a polar non-naturally occurring amino        acid;    -   C²⁷ is absent or S or a polar non-naturally occurring amino        acid; or    -   C²⁸ is absent or R or a basic non-naturally occurring amino        acid;        wherein Formula IV is

-   D¹-D²-D³-D⁴-D⁵-D⁶-D⁷-D⁸-D⁹-D¹⁰-D¹¹-D¹²-D¹³-D¹⁴-D¹⁵-D¹⁶-D¹⁷-D¹⁸-D¹⁹-D²⁰-D²¹-D²²-D²³-D²⁴-D²⁵-D²⁶-D²⁷-D²⁸-D²⁹-D³⁰-D³¹-D³²-D³³-D³⁴-D³⁵-D³⁶-D³⁷-D³⁸-D³⁹-D⁴⁰-D⁴¹-D⁴²-D⁴³-D⁴⁴-D⁴⁵    -   or a pharmaceutically acceptable salt thereof, wherein    -   D¹ is A or a small non-naturally occurring amino acid;    -   D² is A or a small non-naturally occurring amino acid;    -   D³ is Q or a polar non-naturally occurring amino acid;    -   D⁴ is A or a hydrophobic non-naturally occurring amino acid;    -   D⁵ is T or a polar non-naturally occurring amino acid;    -   D⁶ is G or a small non-naturally occurring amino acid;    -   D⁷ is P;    -   D⁸ is L or a hydrophobic non-naturally occurring amino acid;    -   D⁹ is Q or a polar non-naturally occurring amino acid;    -   D¹⁰ is D or a polar non-naturally occurring amino acid;    -   D¹¹ is N or a polar non-naturally occurring amino acid;    -   D¹² is E or a non-naturally occurring amino acid;    -   D¹³ is L or a hydrophobic non-naturally occurring amino acid;    -   D¹⁴ is P;    -   D¹⁵ is G or a small non-naturally occurring amino acid;    -   D¹⁶ is L, or a hydrophobic non-naturally occurring amino acid;    -   D¹⁷ is D or a polar non-naturally occurring amino acid;    -   D¹⁸ is E or a non-naturally occurring amino acid;    -   D¹⁹ is R or a basic non-naturally occurring amino acid;    -   D²⁰ is P;    -   D²¹ is P;    -   D²² is R or a basic non-naturally occurring amino acid;    -   D²³ is A or a small non-naturally occurring amino acid;    -   D²⁴ is H or a basic non-naturally occurring amino acid;    -   D²⁵ is A or a small non-naturally occurring amino acid;    -   D²⁶ is Q or a polar non-naturally occurring amino acid;    -   D²⁷ is H or a basic non-naturally occurring amino acid;    -   D²⁸ is F or a hydrophobic non-naturally occurring amino acid;    -   D²⁹ is H or a basic non-naturally occurring amino acid;    -   D³⁰ is K or a basic non-naturally occurring amino acid;    -   D³¹ is H or a basic non-naturally occurring amino acid;    -   D³² is Q or a polar non-naturally occurring amino acid;    -   D³³ is L or a hydrophobic non-naturally occurring amino acid;    -   D³⁴ is W or a hydrophobic non-naturally occurring amino acid;    -   D³⁵ is P;    -   D³⁶ is S or a polar non-naturally occurring amino acid;    -   D³⁷ is P;    -   D³⁸ is F or a hydrophobic non-naturally occurring amino acid;    -   D³⁹ is R or a basic non-naturally occurring amino acid;    -   D⁴⁰ is A or a hydrophobic non-naturally occurring amino acid;    -   D⁴¹ is L or a hydrophobic non-naturally occurring amino acid;    -   D⁴² is K or a basic non-naturally occurring amino acid;    -   D⁴³ is P;    -   D⁴⁴ is R or a hydrophobic non-naturally occurring amino acid;    -   D⁴⁵ is P;        wherein Formula V is

-   E¹-E²-E³-E⁴-E⁵-E⁶-E⁷-E⁸-E⁹-E¹⁰-E¹¹-E¹²-E¹³-E¹⁴-E¹⁵-E¹⁶-E¹⁷-E¹⁸-E¹⁹-E²⁰-E²¹-E²²-E²³,    or a pharmaceutically acceptable salt thereof, wherein    -   E¹ is A or a small non-naturally occurring amino acid;    -   E² is H or a basic non-naturally occurring amino acid;    -   E³ is A or a small non-naturally occurring amino acid;    -   E⁴ is Q or a polar non-naturally occurring amino acid;    -   E⁵ is H or a basic non-naturally occurring amino acid;    -   E⁶ is F or a hydrophobic non-naturally occurring amino acid;    -   E⁷ is H or a basic non-naturally occurring amino acid;    -   E⁸ is K or a basic non-naturally occurring amino acid;    -   E⁹ is H or a basic non-naturally occurring amino acid;    -   E¹⁰ is Q or a polar non-naturally occurring amino acid;    -   E¹¹ is L or a hydrophobic non-naturally occurring amino acid;    -   E¹² is W or a hydrophobic non-naturally occurring amino acid;    -   E¹³ is P;    -   E¹⁴ is S;    -   E¹⁵ is P;    -   E¹⁶ is F or a hydrophobic non-naturally occurring amino acid;    -   E¹⁷ is R or a basic non-naturally occurring amino acid;    -   E¹⁸ is A or a small non-naturally occurring amino acid.    -   E¹⁹ is L or a hydrophobic non-naturally occurring amino acid;    -   E²⁰ is K or a basic non-naturally occurring amino acid;    -   E²¹ is P;    -   E²² is R or a basic non-naturally occurring amino acid;    -   E²³ is P;        wherein Formula VI is

-   F¹-F²-F³-F⁴-F⁵-F⁶-F⁷-F⁸-F⁹-F¹⁰-F¹¹-F¹²-F¹³-F¹⁴-F¹⁵-F¹⁶-F¹⁷-F¹⁸-F¹⁹-F²⁰-F²¹-F²²-F²³-F²⁴    or a pharmaceutically acceptable salt thereof, wherein    -   F¹ is absent or H or a basic non-naturally occurring amino acid;    -   F² is absent or K or a basic non-naturally occurring amino acid;    -   F³ is absent or R or a basic non-naturally occurring amino acid;    -   F⁴ is T or a polar non-naturally occurring amino acid;    -   F⁵ is I or a hydrophobic non-naturally occurring amino acid;    -   F⁶ is P;    -   F⁷ is M or norleucine (Nle) or another hydrophobic non-naturally        occurring amino acid;    -   F⁸ is F or a hydrophobic non-naturally occurring amino acid;    -   F⁹ is V or a hydrophobic non-naturally occurring amino acid;    -   F¹⁰ is P;    -   F¹¹ is E or a non-naturally occurring amino acid;    -   F¹² is S;    -   F¹³ is T or a polar non-naturally occurring amino acid;    -   F¹⁴ is S;    -   F¹⁵ is K or a basic non-naturally occurring amino acid;    -   F¹⁶ is L or a hydrophobic non-naturally occurring amino acid;    -   F¹⁷ is Q or a polar non-naturally occurring amino acid;    -   F¹⁸ is K or a basic non-naturally occurring amino acid;    -   F¹⁹ is F or a polar non-naturally occurring amino acid;    -   F²⁰ is T or polar non-naturally occurring amino acid;    -   F²¹ is S;    -   F²² is W or a hydrophobic non-naturally occurring amino acid;    -   F²³ is F or a polar non-naturally occurring amino acid;    -   F²⁴ is M or norleucine (Nle) or another hydrophobic        non-naturally occurring amino acid.

The invention in another embodiment includes any one of the foregoingpeptides, wherein said peptide binds to a G-protein coupled receptor(GPCR) protein.

The invention in another embodiment includes any one of the foregoingpeptides, wherein said GPCR protein belongs to the Mas-related family ofproteins, selected from the group consisting of Mas, MrgX1, MrgX2 andother MrgXs; or

wherein said GPCR protein belongs to the FPR-related family of proteins,selected from the group consisting of FPR, FPRL1 and FPRL2.

The invention in another embodiment includes any one of the foregoingpeptides, wherein said peptide activates a GPCR protein.

The invention in another embodiment includes any one of the foregoingpeptides, wherein said GPCR protein is the Mas protein, and when saidpeptide is Formula I or Formula II.

The invention in another embodiment includes any one of the foregoingpeptides, wherein said GPCR protein is the MrgX1 protein (Mas-relatedG-protein coupled receptor member X1, also known as SNSR4), and whensaid peptide is Formula III.

The invention in another embodiment includes any one of the foregoingpeptides, wherein said GPCR protein is the MrgX2 protein (Mas-relatedG-protein coupled receptor member X2), and when said peptide is FormulaI, Formula III, Formula IV, or Formula V.

The invention in another embodiment includes any one of the foregoingpeptides, wherein said GPCR protein is the FPRL1 protein when saidpeptide is Formula III or Formula IV.

The invention in another embodiment includes any one of the foregoingpeptides, wherein said peptide is a degradation product of a naturallyoccurring protein isolated from a cell.

The invention in another embodiment includes any one of the foregoingpeptides, wherein said peptide is isolated from a protein recombinantlyproduced in a cell, selected from a prokaryotic or eukaryotic cell.

The invention in another embodiment includes any one of the foregoingpeptides, wherein said peptide is chemically synthesized in vitro.

The invention in another embodiment includes any one of the foregoingpeptides, wherein said peptide is coupled to a biotin moiety, or whereinsaid peptide includes a disulfide bond, or wherein said peptide is acyclic peptide, or wherein said peptide is a cyclic lactam, or whereinsaid peptide is a branched peptide, or wherein said peptide isphosphorylated, optionally wherein phosphorylation is at an S, T, or Yresidue.

The invention in another embodiment includes any one of the foregoingpeptides, wherein said peptide is modified at its amino terminus,optionally wherein said amino terminal modification includes anN-glycated, N-alkylated, N-acetylated or N-acylated amino acid.

The invention in another embodiment includes any one of the foregoingpeptides, wherein said peptide is pegylated or sialylated.

The invention in another embodiment includes any one of the foregoingpeptides, wherein said peptide includes a C-terminal amidated aminoacid.

The invention in another embodiment includes any one of the foregoingpeptides, wherein said non-naturally occurring amino acid is anomega-amino acid.

The invention in another embodiment includes any one of the foregoingpeptides, wherein said omega-acid is beta-alanine (beta-Ala), or 3aminopropionic (3-aP).

The invention in another embodiment includes any one of the foregoingpeptides, wherein said small non-naturally occurring amino acid issarcosine (Sar), β-alanine (β-Ala), 2,3 diaminopropionic (2,3-diaP) oralpha-aminisobutyric acid (Aib); omega-acid is beta-alanine (beta-Ala),or 3 aminopropionic (3-aP).

The invention in another embodiment includes any one of the foregoingpeptides, wherein said hydrophobic non-naturally occurring amino acid ist butylalanine (t BuA), t butylglycine (t BuG), N methylisoleucine (NMeIle), norleucine (Nle), methylvaline (Mvl), cyclohexylalanine (Cha),phenylglycine (Phg), NaI, β2-thienylalanine (Thi), 2 naphthylalanine (2Nal), or 1,2,3,4-tetrahydroisoquinoline-3 carboxylic acid (Tic).

The invention in another embodiment includes any one of the foregoingpeptides, wherein said basic non-naturally occurring amino acid isornithine (Orn) or homoarginine (Har).

The invention in another embodiment includes any one of the foregoingpeptides, wherein neutral/polar non-naturally occurring amino acid iscitrulline (Cit), Acetyl Lys, or methionine sulfoxide (MSO).

The invention in another embodiment includes any one of the foregoingpeptides, wherein said peptide is less than 75, 50, 30, 20 or 10 aminoacids.

The invention in another embodiment includes any one of the foregoingpeptides, wherein said peptide comprises the amino acid sequence ofFormula I, wherein the peptide is selected from the group consisting of:

-   FLGYCIYLNRKRRGDPAFKRRLRD (SEQ ID NO. 9) monomer or dimer,-   FLGYSIYLNRKRRGDPAFKRRLRD (SEQ ID NO. 10),-   IYLNRKRRGDPAFKRRLRD (SEQ ID NO. 11),-   FAFLGYSIYLNRKRRGDPAF (SEQ ID NO. 12),-   FAFLGYCIYLNRKRRGDPAF (SEQ ID NO. 13) monomer or dimer,-   FAFLGYSIYLN (SEQ ID NO. 18),-   FAFLGYCIYLN (SEQ ID NO. 19) monomer or dimer,-   FAFLGYCIYLNRKRRGDPAFKRRLRD (SEQ ID NO. 20) monomer or dimer,-   FLGYCIYLN (SEQ ID NO. 21) monomer or dimer,-   FLGYCIYLNR (SEQ ID NO. 22) monomer or dimer,-   FLGYCIYLNRKRRGDPAF (SEQ ID NO. 23) monomer or dimer,-   RGDPAF (SEQ ID NO. 24),-   RRGDPAF (SEQ ID NO. 25),-   GDPAFKRRLRD (SEQ ID NO. 28),-   GDPAF (SEQ ID NO. 29),-   IYLN (SEQ ID NO. 30),-   IYLNRKRRGDPAF (SEQ ID NO. 31);    or    wherein the peptide comprises the amino acid sequence of Formula II,    wherein the peptide is selected from the group consisting of:-   monomer or dimer of SMCHRWSRAVLFPAAHRP (SEQ ID NO. 6),-   SMVHRASRAVLFPAAHRP (SEQ ID NO. 7),-   RWSRAVLFPAAHRP (SEQ ID NO. 14),-   HRWSRAVLFPAAHRP (SEQ ID NO. 15),-   WSRAVLFPAAHRP (SEQ ID NO. 16),-   AVLFPAAHRP (SEQ ID NO. 27);    or    wherein the peptide comprises the amino acid sequence of Formula    III, wherein the peptide is selected from the group consisting of:-   GIGSVWHWKHRVATRFTLPRFLQ (SEQ ID NO. 8),-   GIGCVWHWKHRVATRFTLPRFLQ (SEQ ID NO. 39) monomer or dimer,-   GIGCVWHWKHRVATRFTLPRFLQRR (SEQ ID NO. 40) monomer or dimer,-   GIGCVWHWKHRVATRFTLPRFLQRRSS (SEQ ID NO. 41) monomer or dimer,-   GIGCVWHWKHRVATRFTLPRFLQRRSSR (SEQ ID NO. 42) monomer or dimer,-   IGCVWHWKHRVATRFTLPRFLQ (SEQ ID NO. 43) monomer or dimer,-   IGCVWHWKHRVATRFTLPRFLQRR (SEQ ID NO. 44) monomer or dimer,-   IGCVWHWKHRVATRFTLPRFLQRRSS (SEQ ID NO. 45) monomer or dimer,-   IGCVWHWKHRVATRFTLPRFLQRRSSR (SEQ ID NO. 46) monomer or dimer,-   CVWHWKHRVATRFTLPRFLQ (SEQ ID NO. 47) monomer or dimer,-   CVWHWKHRVATRFTLPRFLQRR (SEQ ID NO. 48) monomer or dimer,-   CVWHWKHRVATRFTLPRFLQRRSS (SEQ ID NO. 49) monomer or dimer,-   CVWHWKHRVATRFTLPRFLQRRSSR (SEQ ID NO. 50) monomer or dimer;    or    wherein the peptide comprises the amino acid sequence of Formula IV,    wherein the peptide is selected from the group consisting of:

(SEQ ID NO. 5) AAQATGPLQDNELPGLDERPPRAHAQHFHKHQLWPSPFRALKPRP,(SEQ ID 32) AAQATGPLQDNELPGLDERPP, (SEQ ID NO. 33)AAQATGPLQDNELPGLDERPPRAHAQHFH, (SEQ ID NO. 34)PPRAHAQHFHKHQLWPSPFRALKPRP, (SEQ ID NO. 35) HQLWPSPFRALKPRP;orwherein peptide comprises the amino acid sequence of Formula V, whereinthe peptide is selected from the group consisting of:

AHAQHFHKHQLWPSPFRALKPRP; (SEQ ID NO. 17)orwherein peptide comprises the amino acid sequence of Formula VI, whereinthe peptide is selected from the group consisting of:

TIPMFVPESTSKLQKFTSWFM, (SEQ ID NO. 1) FTSWFM, (SEQ ID NO. 2) LQKFTSWFM,(SEQ ID NO. 3) TIPMFVPESTSTLQKFTSWFM, (SEQ ID NO. 4)HKRTIPMFVPESTSKLQKFTSWFM, (SEQ ID NO. 26) TIPMFVPESTSKLQ,(SEQ ID NO. 36) TIPMFVPESTSTLQ, (SEQ ID NO. 37) TIPMFVPESTS.(SEQ ID NO. 38)

The invention in another embodiment includes any one of the foregoingpeptides, wherein said peptide is conjugated or fused to a secondpeptide or polypeptide, optionally wherein said second peptide orpolypeptide are multiple antigenic peptides (MAP), or wherein saidsecond peptide or polypeptide comprises a portion of an immunoglobulin,or wherein said second peptide or polypeptide comprises albumin or aportion of albumin.

The invention in another embodiment includes any one of the foregoingpeptides, wherein said second peptide or polypeptide includes a signalsequence.

The invention in another embodiment includes any one of the foregoingpeptides, wherein signal sequence comprises: MPSVRSLLRLLAAAAACGAFA (SEQID NO:51) or MPSVRSLLRLLAAAAACGA (SEQ ID NO:52) when said peptide isFormula I; MHWKMLLLLLLYYNAEA (SEQ ID NO:53) when said peptide is FormulaII; MSKSCGNNLAAISVGISLLLLLVVC (SEQ ID NO:54) when said peptide isFormula III; MAHVPARTSPGPGPQLLLLLLPLFLLLLRDVAG (SEQ ID NO: 55) when saidpeptide is Formula IV; MAHVPARTSPGPGPQLLLLLLPLFLLLLRDVAG (SEQ ID NO:55)when said peptide is Formula V; or MATASPSVFLLMVNGQVES (SEQ ID NO:56)when said peptide is Formula VI.

The invention in another embodiment includes a pharmaceuticalcomposition comprising any one of the foregoing peptides and apharmaceutically acceptable carrier.

The invention in another embodiment includes a peptide comprising afragment of any one of the foregoing peptides, wherein said peptidefragment binds or activates a G-protein coupled receptor (GPCR) protein,optionally wherein said GPCR protein belongs to the Mas-related familyof proteins, selected from the group consisting of Mas, MrgX1, MrgX2 andother MrgXs; or wherein said GPCR protein belongs to the FPR-relatedfamily of proteins, selected from the group consisting of FPR, FPRL1 andFPRL2.

The invention in another embodiment includes any one of the foregoingpeptide fragments, wherein said GPCR protein is the Mas protein whensaid peptide is Formula I or II.

The invention in another embodiment includes any one of the foregoingpeptide fragments, wherein said GPCR protein is the MrgX1 protein (alsoknown as SNSR4) when said peptide is Formula III.

The invention in another embodiment includes any one of the foregoingpeptide fragments, wherein said GPCR protein is the MrgX2 protein whensaid peptide is Formula I, Formula III, Formula IV, or Formula V.

The invention in another embodiment includes any one of the foregoingpeptide fragments, wherein said GPCR protein is the FPRL1 protein whensaid peptide is Formula II, Formula IV, or Formula VI.

The invention in another embodiment includes a purified nucleic acidsequence encoding any one of the foregoing peptides.

The invention in another embodiment includes a method of treating adisorder associated with hypertension, said method comprisingadministering to a subject in need thereof a therapeutically effectiveamount of any one of the foregoing peptides, wherein said peptide isFormula I, Formula II, Formula III, Formula IV or Formula V.

The invention in another embodiment includes the foregoing method,wherein said hypertension associated disorder is selected from the groupconsisting of hypertensive heart disease; antihypertension (bloodpressure reduction); systemic and pulmonary high blood pressure;cerebrovascular disease and stroke; heart failure and stroke; leftventricular hypertrophy (LVH); congestive heart failure (CHF);hypertension, high blood pressure; vasodilation; renal hypertension;diuresis; nephritis; natriuresis; scleroderma renal crisis; anginapectoris (stable and unstable); myocardial infarction; heart attack;coronary artery disease; cardiac arrhythmias; atrial fibrillation;portal hypertension; raised intraocular pressure; vascular restenosis;chronic hypertension; valvular disease; myocardial ischemia; acutepulmonary edema; acute coronary syndrome; hypertensive retinopathy;hypertensive pregnancy sickness; preeclampsia; Raynaud's phenomenon;erectile dysfunction and glaucoma. These peptides are also used as avasodilator and in anti-thrombotic therapy.

The invention in another embodiment includes a method of treating acardiovascular disorder, said method comprising administering to asubject in need thereof a therapeutically effective amount of any one ofthe foregoing peptides, wherein said peptide is Formula I, Formula II,Formula IV, or Formula VI.

The invention in another embodiment includes the foregoing method,wherein said cardiovascular disorder is selected from a group consistingof peripheral vascular diseases and coronary artery diseases, myocardialinfarction; heart injury; congestive heart failure (CHF); myocardialfailure; myocardial hypertrophy; ischemic cardiomyopathy; systolic heartfailure; diastolic heart failure; stroke; thrombotic stroke; concentricLV hypertrophy, myocarditis; cardiomyopathy; hypertrophiccardiomyopathy; myocarditis; decompensated heart failure; ischemicmyocardial disease; congenital heart disease; angina pectoris;prevention of heart remodeling or ventricular remodeling aftermyocardial infarction; ischemia—reperfusion injury in ischemic andpost-ischemic events (e.g. myocardial infarct); cerebrovascularaccident; mitral valve regurgitation; hypertension; hypotension;restenosis; fibrosis; thrombosis; and platelet aggregation.

The invention in another embodiment includes a method of treating anischemia-reperfusion injury related disorder, said method comprisingadministering to a subject in need thereof a therapeutically effectiveamount of anyone of foregoing peptides, wherein said peptide is FormulaI, Formula II, Formula IV, or Formula VI.

The invention in another embodiment includes the foregoing method,wherein said ischemia-reperfusion injury related disorder is associatedwith ischemic and post-ischemic events in organs and tissues, and thedisorder is selected from a group consisting of thrombotic stroke;myocardial infarction; angina pectoris; embolic vascular occlusions;peripheral vascular insufficiency; splanchnic artery occlusion; arterialocclusion by thrombi or embolisms, arterial occlusion by non-occlusiveprocesses such as following low mesenteric flow or sepsis; mesentericarterial occlusion; mesenteric vein occlusion; ischemia-reperfusioninjury to the mesenteric microcirculation; ischemic acute renal failure;ischemia-reperfusion injury to the cerebral tissue; intestinalintussusception; hemodynamic shock; tissue dysfunction; organ failure;restenosis; atherosclerosis; thrombosis; platelet aggregation; orfollowing conditions selected from a list comprising of procedures suchas cardiac surgery; organ surgery; organ transplantation; angiography;cardiopulmonary and cerebral resuscitation.

The invention in another embodiment includes a method of treating acentral nervous system (CNS) disorder or a peripheral nervous system(PNS) disorder in a subject, said method comprising administering to asubject in need thereof a therapeutically effective amount of anyone offoregoing peptides, and wherein said peptide is Formula I, Formula II,Formula III, Formula IV, or Formula V.

The invention in another embodiment includes the foregoing method,wherein said CNS or PNS disorder is selected from the group consistingof central and peripheral degenerative neuropathies; neuroprotection;impaired cognition; anxiety disorders, pain control, food intake, abehavioral disorder, a learning disorder, a sleep disorder, a memorydisorder, a pathologic response to anesthesia, addiction, depression,migraine, a menstruation disorder, muscle spasm, opiate dependence,dementia, Alzheimer's disease, Parkinson's disease, cortical function,and locomotor activity.

The invention in another embodiment includes a method of treating aninflammatory disorder in a subject, said method comprising administeringto a subject in need thereof a therapeutically effective amount ofanyone of the foregoing peptides, wherein said peptide is Formula I,Formula II, Formula III, Formula IV, Formula V, or Formula VI.

The invention in another embodiment includes the foregoing method,wherein said inflammatory disorder is selected from the group consistingof gastritis, gout, gouty arthritis, arthritis, rheumatoid arthritis,inflammatory bowel disease, Crohn's disease, ulcerative colitis, ulcers,chronic bronchitis, asthma, allergy, acute lung injury, pulmonaryinflammation, airway hyper-responsiveness, vasculitis, septic shock andinflammatory skin disorders, selected from the list comprising ofpsoriasis, atopic dermatitis, and eczema.

The invention in another embodiment includes a method of treatinginflammatory conditions associated with an infection, said infectionbeing a bacterial infection or viral infection or an infection caused byanother type of pathogen, in a subject, said method comprisingadministering to a subject in need thereof a therapeutically effectiveamount of anyone of the foregoing peptides, wherein said peptide isFormula I, Formula II, Formula IV, or Formula VI.

The invention in another embodiment includes the foregoing method,wherein said inflammatory disorder is associated with a bacterialinfection or viral infection or an infection caused by another type ofpathogen, selected from a group consisting of a viral infection causedby human immunodeficiency virus I (HIV-1) or HIV-2, acquired immunedeficiency (AIDS), West Nile encephalitis virus, coronavirus,rhinovirus, influenza virus, dengue virus, hemorrhagic fever; anotological infection; severe acute respiratory syndrome (SARS), sepsisand sinusitis.

The invention in another embodiment includes a method of treating ametabolic disorder in a subject, said method comprising administering toa subject in need thereof a therapeutically effective amount of anyoneof the foregoing peptides, wherein said peptide is Formula I, FormulaII, Formula IV, or Formula VI.

The invention in another embodiment includes the foregoing method,wherein said metabolic disorder is selected from a group consisting ofdiabetes, diabetis mellitus, lipodystrophy, hyperthyroidism, glaucoma,hyperlipidaemia, non-insulin dependent diabetes, appetite control andobesity.

The invention in another embodiment includes a method of treating afibrotic condition in a subject, involving tissue remodeling followinginflammation or ischemia-reperfusion injury, said method comprisingadministering to a subject in need thereof a therapeutically effectiveamount of anyone of the foregoing peptides, wherein said peptide isFormula I, Formula II, Formula III, Formula IV, Formula V or Formula VI.

The invention in another embodiment includes the foregoing method,wherein said fibrotic conditions is selected from a group consisting ofendomyocardial fibrosis; mediastinal fibrosis; idiopathy pulmonaryfibrosis; pulmonary fibrosis; retroperitoneal fibrosis; fibrosis of thespleen; fibrosis of the pancreas; hepatic fibrosis (cirrhosis);fibromatosis; granulomatous lung disease; and glomerulonephritis

The invention in another embodiment includes a method of prevention andtreatment of a disease in a subject, involving reduction of oxygenreactive species with consequent endothelial dysfunction, said methodcomprising administering to a subject in need thereof a therapeuticallyeffective amount of anyone of the foregoing peptides, wherein saidpeptide is Formula I, or Formula II.

The invention in another embodiment includes the foregoing method,wherein said endothelial dysfunction disease is selected from a groupconsisting of cardiovascular diseases, high blood pressure,atherosclerosis, thrombosis, myocardial infarct, heart failure, renaldiseases, plurimetabolic syndrome, erectile dysfunction; vasculitis; anddiseases of the central nervous system (CNS).

The invention in another embodiment includes a method of treating arespiratory disease in a subject, said method comprising administeringto a subject in need thereof a therapeutically effective amount ofanyone of the foregoing peptides, wherein said peptide is Formula I,Formula II, Formula IV, or Formula VI.

The invention in another embodiment includes the foregoing method,wherein said respiratory disease is selected from a group consisting ofasthma, bronchial disease, lung diseases, cystic fibrosis, chronicobstructive pulmonary disease (COPD), Acute Respiratory DistressSyndrome (ARDS), severe acute respiratory syndrome (SARS).

The invention in another embodiment includes a method of preventing ortreating a skin injury or tissue repair, said method comprisingadministering to a subject in need thereof a therapeutically effectiveamount of anyone of the foregoing peptides, wherein said peptide isFormula I, Formula II, Formula IV, or Formula VI.

The invention in another embodiment includes the foregoing method,wherein said skin injury is selected from a group selected of dermalrepair, wound healing; burns, erythemas, skin or tissue lesions, andskin tumors.

The invention in another embodiment includes a method of treating a bonedisease in a subject, said method comprising administering to a subjectin need thereof a therapeutically effective amount of anyone of theforegoing peptides, wherein said peptide is Formula II, Formula IV, orFormula VI.

The invention in another embodiment includes the foregoing method,wherein said bone disease is osteoporosis.

The invention in another embodiment includes a method of treating aurogenital disorder or a genitor-urological disorder in a subject, saidmethod comprising administering to a subject in need thereof atherapeutically effective amount of anyone of the foregoing peptides,wherein said peptide is Formula I, Formula II.

The invention in another embodiment includes the foregoing method,wherein said urogenital disorder or genitor-urological disorders isselected from group consisting of a renal disease; a bladder disorder;disorders of the reproductive system; gynecologic disorders; urinarytract disorder; incontinence; disorders of the male (spermatogenesis,spermatic motility), and female reproductive system; sexual dysfunction;erectile dysfunction; embryogenesis; pregnancy related disorders andpregnancy monitoring.

The invention in another embodiment includes a method of activating orinducing chemoattraction of blood cells to a site of injury in asubject, said method comprising administering to a subject in needthereof a therapeutically effective amount of anyone of the foregoingpeptides, wherein said peptide is Formula II, Formula IV or Formula VI.

The invention in another embodiment includes the foregoing method,wherein said blood cells are selected from a group consisting ofphagocyte cells, platelets, monocytes, macrophages, neutrophils,eosinophils and lymphocytes.

The invention in another embodiment includes a method of treating acytopenia in a subject, said method comprising administering to asubject in need thereof a therapeutically effective amount of anyone ofthe foregoing peptides, wherein said peptide is Formula I, Formula II,Formula IV, or Formula VI.

The invention in another embodiment includes the foregoing method,wherein said cytopenia is selected from a group consisting ofmultilineage cytopenia, a thrombocytopenia, anemia, anemia due to renalfailure; lymphopenia, leucopenia, neutropenia,radio/chemotherapy-related neutropenia; and platelet disorders.

The invention in another embodiment includes a method of treating animmune related disorder in a subject, said method comprisingadministering to a subject in need thereof a therapeutically effectiveamount of anyone of the foregoing peptides, wherein said peptide isFormula I, Formula II, Formula IV, or Formula VI.

The invention in another embodiment includes the foregoing method,wherein said immune related disorder is selected from a group consistingof graft versus host disease; transplant rejection, bone marrowtransplantation.

The invention in another embodiment includes the foregoing method,wherein said immune related disorder is an autoimmune disease and isselected from a group consisting of multiple sclerosis, psoriasis,rheumatoid arthritis, systemic lupus erythematosus, ulcerative colitis,Crohn's disease, transplant rejection, immune disorders associated withgraft transplantation rejection, benign lymphocytic angiitis, lupuserythematosus, Hashimoto's thyroiditis, primary myxedema, Graves'disease, pernicious anemia, autoimmune atrophic gastritis, Addison'sdisease, insulin dependent diabetes mellitis, Good pasture's syndrome,myasthenia gravis, pemphigus, sympathetic ophthalmia, autoimmuneuveitis, autoimmune hemolytic anemia, idiopathic thrombocytopenia,primary biliary cirrhosis, chronic action hepatitis, ulceratis colitis,Sjogren's syndrome, rheumatic disease, polymyositis, scleroderma, mixedconnective tissue disease, inflammatory rheumatism, degenerativerheumatism, extra-articular rheumatism, collagen diseases, chronicpolyarthritis, psoriasis arthropathica, ankylosing spondylitis, juvenilerheumatoid arthritis, periarthritis humeroscapularis, panarteriitisnodosa, progressive systemic scleroderma, arthritis uratica,dermatomyositis, muscular rheumatism, myositis, myogelosis andchondrocalcinosis.

The invention in another embodiment includes a method of treating acancer or inflammation associated with cancer in a patient, said methodcomprising administering to a subject in need thereof a therapeuticallyeffective amount of anyone of the foregoing peptides, wherein saidpeptide is Formula I, Formula II, Formula IV or Formula VI.

The invention in another embodiment includes the foregoing method,wherein said cancer is selected from a group consisting of colon cancer,lung cancer, breast cancer, prostate cancer, brain cancer, pancreaticcancer, ovarian cancer, kidney cancer, melanoma, glioma, a carcinoma, asarcoma, a leukemia, or lymphoma, including their invasive andmetastatic forms.

The invention in another embodiment includes a method of treating orcontrolling pain in a patient, said method comprising administering to asubject in need thereof a therapeutically effective amount of anyone ofthe foregoing peptides wherein said peptide is Formula I, Formula III,Formula IV, or Formula V.

The invention in another embodiment includes the foregoing method,wherein said pain is selected from a group consisting of complexregional pain, muscoskeletal pain, neuropathic pain, post-herpetic pain,pain associated with cancer, or post-operative pain.

The invention in another embodiment includes a method of treating akidney disease in a patient, said method comprising administering to asubject in need thereof a therapeutically effective amount of anyone ofthe foregoing peptides, wherein said peptide is Formula I, Formula II,Formula III, Formula IV or Formula V.

The invention in another embodiment includes the foregoing method,wherein said kidney diseases is selected from a group consisting ofdiabetic nephropathy; glomerulosclerosis; nephropathies; renalimpairment; scleroderma renal crisis and chronic renal failure.

The invention in another embodiment includes a method of treating ablood disease in a patient, said method comprising administering to asubject in need thereof a therapeutically effective amount of anyone ofthe foregoing peptides, wherein said peptide is Formula I, or FormulaII.

The invention in another embodiment includes the foregoing method,wherein said blood disease is selected from a group consisting ofangioplasty (endoluminal prosthesis and post angioplasty restenosis);haematopoiesis; erythrocytosis; and disorders of the blood crasis, suchas post radiotherapy.

The invention in another embodiment includes a method of treating anangiogenesis related disease in a patient, said method comprisingadministering to a subject in need thereof a therapeutically effectiveamount of anyone of the foregoing peptides, wherein said peptide isFormula I, Formula II, Formula IV or Formula VI.

The invention in another embodiment includes the foregoing method,wherein said angiogenesis related disease is retinal angiogenesis inhuman ocular diseases and is selected from a group consisting ofdiabetes mellitus, retinopathy of prematury, and age-related maculardegeneration.

The invention in another embodiment includes the foregoing method,wherein said angiogenesis related disease is primary or metastaticcancer, and is selected from a group consisting of prostate cancer,brain cancer, breast cancer, colorectal cancer, lung cancer, ovariancancer, pancreatic cancer, renal cancer, cervical cancer, melanoma, softtissue sarcomas, lymphomas, head-and-neck cancer, and glioblastomas.

The invention in another embodiment includes a method of treating agenetic polymorphism consequent diseases in a patient, said methodcomprising administering to a subject in need thereof a therapeuticallyeffective amount of anyone of the foregoing peptides, wherein saidpeptide is Formula I, or Formula II.

The invention in another embodiment includes the foregoing method,wherein said genetic polymorphism consequent diseases is selected from agroup consisting of DD type of the angiotensin converting enzyme; type Iand type II diabetes mellitus and complications; diabetic mellitusprophylaxis; diabetic maculopathy; and diabetic nephropathy.

The invention in another embodiment includes a method of preventing ortreating a organic alterations produced by aging in a patient, saidmethod comprising administering to a subject in need thereof atherapeutically effective amount of anyone of the foregoing peptides,wherein said peptide is Formula I, or Formula II.

The invention in another embodiment includes a method of preventing ortreating alopecia in a patient, including chemotherapy (such asetoposide)-induced alopecia, said method comprising administering to asubject in need thereof a therapeutically effective amount of anyone ofthe foregoing peptides, wherein said peptide is Formula I, Formula II,Formula IV or Formula VI.

The invention in another embodiment includes a method of preventing ortreating diseases that involve alterations in the musculardifferentiation, maturation and regeneration in muscular atrophies in apatient, said method comprising administering to a subject in needthereof a therapeutically effective amount of anyone of the foregoingpeptides, wherein said peptide is Formula I, or Formula II.

The invention in another embodiment includes the foregoing method,wherein said muscular alteration or atrophy is selected from a groupconsisting of cachexia; prolonged restriction to bed due to numerousfactors; chronic use of corticoids; and varied neurological syndromes,traumatisms and degenerative diseases that lead to muscular atrophy.

The invention in another embodiment includes the cDNA that encodes thepeptide sequences of the invention, which can be used in gene therapy.

If desired, gene therapy can be used to deliver to a subject a peptideaccording to the invention. A nucleic acid encoding the peptide can beinserted into vectors, which are then used as gene therapy vectors. Genetherapy vectors can be delivered to a subject by, for example,intravenous injection, local administration or by stereotacticinjection. The pharmaceutical preparation of the gene therapy vector caninclude the gene therapy vector in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery vector can beproduced intact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells that producethe gene delivery system.

The invention in another embodiment includes combination therapy usingone or more peptides of the present invention provided in combinationwith another therapeutic agent or agents. As used herein, the term“combination therapy” refers to treatment of a single condition ordisease involving the concomitant use of more than one therapeuticagent.

The invention in another embodiment includes an antibody thatselectively binds to an epitope in anyone of the foregoing peptides.

The invention in another embodiment includes anyone of the foregoingantibodies, wherein said peptide is

-   monomer or dimer of FLGYCIYLNRKRRGDPAFKRRLRD (SEQ ID NO. 9),-   FLGYSIYLNRKRRGDPAFKRRLRD (SEQ ID NO. 10),-   IYLNRKRRGDPAFKRRLRD (SEQ ID NO. 11),-   FAFLGYSIYLNRKRRGDPAF (SEQ ID NO. 12),-   monomer or dimer of FAFLGYCIYLNRKRRGDPAF , (SEQ ID NO. 13),-   FAFLGYSIYLN (SEQ ID NO. 18),-   monomer or dimer of FAFLGYCIYLN (SEQ ID NO. 19),-   monomer or dimer of FAFLGYCIYLNRKRRGDPAFKRRLRD (SEQ ID NO. 20),-   monomer or dimer of FLGYCIYLN (SEQ ID NO. 21),-   monomer or dimer of FLGYCIYLNR (SEQ ID NO. 22),-   monomer or dimer of FLGYCIYLNRKRRGDPAF (SEQ ID NO. 23),-   RGDPAF (SEQ ID NO. 24),-   RRGDPAF (SEQ ID NO. 25),-   GDPAFKRRLRD (SEQ ID NO. 28),-   GDPAF (SEQ ID NO. 29),-   IYLN (SEQ ID NO. 30),-   IYLNRKRRGDPAF (SEQ ID NO. 31),-   monomer or dimer of SMCHRWSRAVLFPAAHRP (SEQ ID NO. 6),-   SMVHRWSRAVLFPAAHRP (SEQ ID NO. 7)-   RWSRAVLFPAAHRP (SEQ ID NO. 14),-   HRWSRAVLFPAAHRP (SEQ ID NO. 15),-   WSRAVLFPAAHRP (SEQ ID NO. 16),-   AVLFPAAHRP (SEQ ID NO. 27),-   GIGSVWHWKHRVATRFTLPRFLQ (SEQ ID NO. 8),-   monomer or dimer of GIGCVWHWKHRVATRFTLPRFLQ (SEQ ID NO. 39),-   monomer or dimer of GIGCVWHWKHRVATRFTLPRFLQRR (SEQ ID NO. 40),-   monomer or dimer of GIGCVWHWKHRVATRFTLPRFLQRRSS (SEQ ID NO. 41),-   monomer or dimer of GIGCVWHWKHRVATRFTLPRFLQRRSSR (SEQ ID NO. 42),-   monomer or dimer of IGCVWHWKHRVATRFTLPRFLQ (SEQ ID NO. 43),-   monomer or dimer of IGCVWHWKHRVATRFTLPRFLQRR (SEQ ID NO. 44),-   monomer or dimer of IGCVWHWKHRVATRFTLPRFLQRRSS (SEQ ID NO. 45),-   monomer or dimer of IGCVWHWKHRVATRFTLPRFLQRRSSR (SEQ ID NO. 46),-   monomer or dimer of CVWHWKHRVATRFTLPRFLQ (SEQ ID NO. 47),-   monomer or dimer of CVWHWKHRVATRFTLPRFLQRR (SEQ ID NO. 48),-   monomer or dimer of CVWHWKHRVATRFTLPRFLQRRSS (SEQ ID NO. 49),-   monomer or dimer of CVWHWKHRVATRFTLPRFLQRRSSR (SEQ ID NO. 50),-   AAQATGPLQDNELPGLDERPPRAHAQHFHKHQLWPSPFRALKPRP (SEQ ID NO. 5),-   AAQATGPLQDNELPGLDERPP (SEQ ID 32),-   AAQATGPLQDNELPGLDERPPRAHAQHFH (SEQ ID NO. 33),-   PPRAHAQHFHKHQLWPSPFRALKPRP (SEQ ID NO. 34),-   HQLWPSPFRALKPRP (SEQ ID NO. 35),-   AHAQHFHKHQLWPSPFRALKPRP (SEQ ID NO. 17),-   TIPMFVPESTSKLQKFTSWFM (SEQ ID NO. 1),-   FTSWFM (SEQ ID NO. 2),-   LQKFTSWFM (SEQ ID NO. 3),-   TIPMFVPESTSTLQKFTSWFM (SEQ ID NO. 4),-   HKRTIPMFVPESTSKLQKFTSWFM (SEQ ID NO. 26),-   TIPMFVPESTSKLQ (SEQ ID NO. 36),-   TIPMFVPESTSTLQ (SEQ ID NO. 37),-   TIPMFVPESTS (SEQ ID NO. 38).

The invention in another embodiment includes anyone of the foregoingantibodies, wherein the antibody is a monoclonal antibody.

The invention in another embodiment includes anyone of the foregoingantibodies, wherein the antibody is conjugated or coupled to adetectable label, a radioactive label, an enzyme, a fluorescent label, aluminescent label, a bioluminescent label, or a therapeutic agent.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the invention, suitable methods and materials aredescribed below. All publications, patent applications, patents, andother references mentioned herein are incorporated by reference in theirentirety. In the case of conflict, the present Specification, includingdefinitions, will control. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 lists the peptides described in this application.

FIG. 2 is a line graph demonstrating the effect of Peptide 60_S oncalcium flux in CHO-K1 cells transfected with MrgX1.

FIG. 3 is a line graph demonstrating the dose response of CHO-K1 cellstransfected with MrgX1 to Peptide 60_S.

FIG. 4 is a line graph demonstrating the effect of Peptide 60_S oncalcium flux in CHO-K1 cells transfected with MrgX2.

FIG. 5 is a line graph demonstrating the effect of Peptide 94 on calciumflux in CHO-K1 cells transfected with MrgX2.

FIG. 6 is a line graph demonstrating the effect of Peptide 61_S oncalcium flux in CHO-K1 cells transfected with MrgX2.

FIG. 7 is a line graph demonstrating the effect of Peptide 63 on calciumflux in CHO-K1 cells transfected with MrgX2.

FIG. 8 is a line graph showing the effect of Peptide 33_V on calciumflux in CHO-K1 cells transfected with Mas.

FIG. 9 is a line graph demonstrating the dose response of CHO-K1 cellstransfected with Mas to Peptides P33_V, P33_mono, P33_dimer, andP33_(—)5.

FIG. 10 is a line graph demonstrating the dose response of CHO-K1 cellstransfected with Mas to Peptides P33_(—)8, P33_(—)9, P33_(—)10, andP33_V.

FIG. 11 is a line graph illustrating the effect of Peptide 61_S oncalcium flux in CHO-K1 cells transfected with Mas).

FIG. 12 is a line graph demonstrating the dose response of CHO-K1 cellstransfected with Mas to P61_S, P61_mono, and P61_dimer.

FIG. 13 is a line graph demonstrating the dose response of CHO-K1 cellstransfected with Mas to P61_(—)4, P61_(—)11S, and P61_S.

FIG. 14 is a line graph demonstrating the effect of Peptide 60_S oncalcium flux in CHO-K1 cells transfected with FPRL1.

FIG. 15 is a line graph demonstrating the effect of Peptide 33_V oncalcium flux in CHO-K1 cells transfected with FPRL1.

FIG. 16 is a line graph showing the effect of Peptide 33 and itsderivatives on calcium flux in CHO-K1 cells transfected with FPRL1, ascompared to W peptide.

FIG. 17 is a line graph demonstrating the effect of Peptide 94 oncalcium flux in CHO-K1 cells transfected with FPRL1.

FIG. 18 is a line graph demonstrating the effect of Peptide 58 oncalcium flux in CHO-K1 cells transfected with FPRL1.

FIG. 19 is a line graph demonstrating the effect of Peptide P58 and someof its derivatives on calcium flux in CHO-K1 cells transfected withFPRL1, as compared to W peptide.

FIG. 20 is a line graph demonstrating the effect of Peptide P58 and someof its derivatives on calcium flux in CHO-K1 cells transfected withFPRL1, as compared to W peptide.

FIG. 21 is a line graph demonstrating the effect of Peptide 58 and itsderivatives on calcium flux in CHO-K1 cells transfected with FPRL1, ascompared to Ac2-26.

FIG. 22 is a response curve depicting the results of a competitiveradioligand binding assay for Peptide 58 to FPRL-1.

FIG. 23 is a table showing the effect of P58 and Ac2-26 onzymosan-induced leukocyte influx to air pouch cavities, at the 4 htime-point.

FIG. 24 is a table depicting the effect of P58 and Ac2-26 onzymosan-induced neutrophil (GR-1⁺ cells) influx into air pouch cavities,at the 4h time-point.

FIG. 25 is a representative flow cytometry histogram for GR-1 stainingHistograms demonstrate a representative staining from one mouse fromeach of the experimental groups. Grey Line: non specific backgroundstaining with IgG labeled with phycoerithrin. Black Histograms showGR-1⁺ staining. The numbers above the black histograms show thepercentage of GR-1⁺ events in the leukocyte population.

FIG. 26 is a table demonstrating the effect of P58 and analogs onzymosan-induced leukocyte influx to air pouch cavities at the 4htime-point.

FIG. 27 is a table showing the effect of P58 and analogs on zymosaninduced neutrophil (GR-1⁺) influx to air pouch cavities at the 4 htime-point.

FIG. 28 is a representative scheme of the experiment flow showing thecumulative concentration response curve with P61_S, P61_D, P33_V, andP33_D in vessels pre-contracted with 0.1 uM phenylephrine (PHE). The Xaxis represents the time and the y axis depicts the level ofcontraction.

FIG. 29 is a graph demonstrating the vasodilator effect of P61_S inaortic rings from Wistar rat containing (E+) or lacking functionalendothelium (E−). Each point represents the mean±SEM generated from atleast 6 separated experiments ***p<0.001.

FIG. 30 is a graph depicting the vasodilator effect of P61_S in aorticrings from Wistar rat in the absence (control) or presence of L-NAME, aNO synthase inhibitor. Each point represents the mean±SEM generated fromat least 5 separated experiments ***p<0.001.

FIG. 31 is a graph showing the vasodilator effect of P61_D in aorticrings from Wistar rat containing (E+) or lacking functional endothelium(E−). Each point represents the mean±SEM generated from at least 8separated experiments **p<0.01.

FIG. 32 is a graph illustrating the vasodilator effect of P61_S inaortic rings from Wistar rat in the absence (control) or presence ofL-NAME. Each point represents the mean±SEM generated from at least 5separated experiments *p<0.05; ***p<0.001.

FIG. 33 is a graph demonstrating the vasodilator effect of P33_V inaortic rings from Wistar rat containing (E+) or lacking functionalendothelium (E−). Each point represents the mean±SEM generated from atleast 6 separated experiments **p<0.01.

FIG. 34 is a graph depicting the vasodilator effect of P33_V in aorticrings from Wistar rat in the absence (control) or presence of L-NAME.Each point represents the mean±SEM generated from at least 6 separatedexperiments **p<0.01.

FIG. 35 is a graph showing the vasodilator effect of P33_D in aorticrings from Wistar rat containing (E+) or lacking functional endothelium(E−). Each point represents the mean±SEM generated from at least 6separated experiments ***p<0.001.

FIG. 36 is a graph illustrating the vasodilator effect of P33_D inaortic rings from Wistar rat in the absence (control) or presence ofL-NAME. Each point represents the mean±SEM generated from at least 5separated experiments *p<0.05.

FIG. 37 is a graph showing the vasodilator effect of Ang-(1-7) in aorticrings from Wistar rat containing (E+) or lacking functional endothelium(E−). Each point represents the mean±SEM generated from at least 4separated experiments *p<0.05.

FIG. 38 is a graph demonstrating the vasodilator effect of Ang-(1-7) inaortic rings from Wistar rat in the absence (control) or presence ofL-NAME. Each point represents the mean±SEM generated from at least 5separated experiments ***p<0.001.

FIG. 39 is a line graph demonstrating the dose response of CHO-K1 cellstransfected with Mas to P33_V and P61_S.

FIG. 40 is a histogram depicting Fibronectin deposition in Isoproterenolinduced rats in control animals and in the presence of P61_D.

FIG. 41 demonstrates Fibronectin deposition in Isoproterenol inducedrats in the presence or absence of P61_S.

FIG. 42 demonstrates Collagen III deposition in Isoproterenol inducedrats in the presence or absence of P61_D.

FIG. 43 demonstrates Collagen III deposition in Isoproterenol inducedrats in the presence or absence of P61_S.

FIG. 44 demonstrates Collagen I deposition in Isoproterenol induced ratsin the presence or absence of P61_S.

FIG. 45 demonstrates Collagen I deposition in Isoproterenol induced ratsin the presence or absence of P61_D.

FIG. 46 demonstrates a ratio of left ventricular mass to body weight incontrol and Isoproterenol treated rats in the presence or absence ofLosartan.

FIG. 47 demonstrates a ratio of left ventricular mass to body weight incontrol and Isoproterenol treated rats in the presence or absence ofP33_V.

FIG. 48 ratio of left ventricular mass to body weight in control andIsoproterenol treated rats in the presence or absence of P61_S.

FIG. 49 demonstrates a ratio of left ventricular mass to body weight incontrol and Isoproterenol treated rats in the presence or absence ofP33_D.

FIG. 50 demonstrates a ratio of left ventricular mass to body weight incontrol and Isoproterenol treated rats in the presence or absence ofP61_D.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides bioactive peptides. These peptides are useful,inter alia, for treating a variety of indications and disorders, whichare discussed in detail below. In some embodiments, the peptides areligands for GPCR receptors.

Provided are bioactive peptides falling within Formula I (also known asthe Peptide 61-type peptides), Formula II (Peptide 33-type peptides),Formula III (Peptide 60-type peptides), Formula IV (also known as thePeptide 94-type peptides), Formula V (Peptide 63-type peptides), orFormula VI (Peptide 58-type peptides).

Formula I includes compounds falling within the following formula:

-   A¹-A²-A³-A⁴-A⁵-A⁶-A⁷-A⁸-A⁹-A¹⁰-A¹¹-A^(l2)-A¹³-A¹⁴-A¹⁵-A¹⁶-A¹⁷-A¹⁸-A¹⁹-A²⁰-A²¹-A²²-A²³-A²⁴-A²⁵-A²⁶,    -   or a pharmaceutically acceptable salt thereof, wherein    -   A¹ is absent or F or a hydrophobic non-naturally occurring amino        acid;    -   A² is absent or A or a small non-naturally occurring amino acid;    -   A³ is absent or F or a hydrophobic non-naturally occurring amino        acid;    -   A⁴ is absent or L or a hydrophobic non-naturally occurring amino        acid;    -   A⁵ is absent or G or a small non-naturally occurring amino acid;    -   A⁶ is absent or Y or a hydrophobic non-naturally occurring amino        acid;    -   A⁷ is absent or S or C;    -   A⁸ is absent or I or a hydrophobic non-naturally occurring amino        acid;    -   A⁹ is absent or Y or a hydrophobic non-naturally occurring amino        acid;    -   A¹⁰ is absent or L or a hydrophobic non-naturally occurring        amino acid;    -   A¹¹ is absent or N or a polar non-naturally occurring amino        acid;    -   A^(l2) is absent or R or a basic non-naturally occurring amino        acid;    -   A¹³ is absent or K or a basic non-naturally occurring amino        acid;    -   A¹⁴ is absent or R or a basic non-naturally occurring amino        acid;    -   A¹⁵ is absent or R or a basic non-naturally occurring amino        acid;    -   A¹⁶ is absent or G, or a small non-naturally occurring amino        acid;    -   A¹⁷ is absent or D or a polar non-naturally occurring amino        acid;    -   A¹⁸ is absent or P;    -   A¹⁹ is absent or A or a hydrophobic non-naturally occurring        amino acid;    -   A²⁰ is absent or F or a hydrophobic non-naturally occurring        amino acid;    -   A²¹ is absent or K or a basic non-naturally occurring amino        acid;    -   A²² is absent or R or a basic non-naturally occurring amino        acid;    -   A²³ is absent or R or a basic non-naturally occurring amino        acid;    -   A²⁴ is absent or L or a hydrophobic non-naturally occurring        amino acid;    -   A²⁵ is absent or R or a hydrophobic non-naturally occurring        amino acid;    -   A²⁶ is absent or D or a polar non-naturally occurring amino        acid.

In some embodiments, a peptide of Formula I includes the amino acidsequences FLGYCIYLNRKRRGDPAFKRRLRD monomer or dimer (SEQ ID NO:9), alsoreferred to herein is as Peptide 61 (P61), or FLGYSIYLNRKRRGDPAFKRRLRD(SEQ ID NO:10), also referred to herein is as Peptide 61_S (P61_S).

P61-type peptides falling within Formula I are listed in Table 1 below:

Table 1: P61-type peptides falling within Formula I.

Name Sequence SEQ ID P_61 FLGYCIYLNRKRRGDPAFKRRLRD SEQ ID 9 P_61_SFLGYSIYLNRKRRGDPAFKRRLRD SEQ ID 10 P61_4 IYLNRKRRGDPAFKRRLRD SEQ ID 11P61_11S FAFLGYSIYLNRKRRGDPAF SEQ ID 12 P61_11 FAFLGYCIYLNRKRRGDPAFSEQ ID 13 P61_5S FAFLGYSIYLN SEQ ID 18 P61_5 FAFLGYCIYLN SEQ ID 19P61 derivative FAFLGYCIYLNRKRRGDPAFKRRLRD SEQ ID 20 P61 derivativeFLGYCIYLN SEQ ID 21 P61 derivative FLGYCIYLNR SEQ ID 22 P61 derivativeFLGYCIYLNRKRRGDPAF SEQ ID 23 P61 derivative RGDPAF SEQ ID 24P61 derivative RRGDPAF SEQ ID 25 P61_14 GDPAFKRRLRD SEQ ID 28 P61_16GDPAF SEQ ID 29 P61_7 IYLN SEQ ID 30 P61_13 IYLNRKRRGDPAF SEQ ID 31

Compounds of Formula II include the following:

-   B¹-B²-B³-B⁴-B⁵-B⁶-B⁷-B⁸-B⁹-B¹⁰-B¹¹-B¹²-B¹³-B¹⁴-B¹⁵-B¹⁶-B¹⁷-B¹⁸, or a    pharmaceutically acceptable salt thereof, wherein    -   B¹ is absent or S;    -   B² is absent or M or norleucine (Nle) or another hydrophobic        non-naturally occurring amino acid;    -   B³ is absent or C or V;    -   B⁴ is absent or H or a basic non-naturally occurring amino acid;    -   B⁵ is absent or R or a basic non-naturally occurring amino acid;    -   B⁶ is absent or W or a hydrophobic non-naturally occurring amino        acid    -   B⁷ is absent or S;    -   B⁸ is absent or R or a hydrophobic non-naturally occurring amino        acid;    -   B⁹ is A or a small non-naturally occurring amino acid;    -   B¹⁰ is V or a hydrophobic non-naturally occurring amino acid;    -   B¹¹ is L or a hydrophobic non-naturally occurring amino acid;    -   B¹² is F or a hydrophobic non-naturally occurring amino acid;    -   B¹³ is P;    -   B¹⁴ is A or a hydrophobic non-naturally occurring amino acid;    -   B¹⁵ is A or a hydrophobic non-naturally occurring amino acid;    -   B¹⁶ is H or a basic non-naturally occurring amino acid;    -   B¹⁷ is R or a basic non-naturally occurring amino acid;    -   B¹⁸ is P.

Examples of peptides within Formula II include SMCHRWSRAVLFPAAHRPmonomer or dimer (SEQ ID NO:6), also referred herein as P33; andSMVHRWSRAVLFPAAHRP (SEQ ID NO:7), also referred herein as P33_V. Otherexamples of P33-type peptides falling within Formula II are listed inTable 2 below:

Table 2: P33-type peptides falling within Formula II.

Name Sequence SEQ ID P33 SMCHRWSRAVLFPAAHRP SEQ ID 6 P33_VSMVHRWSRAVLFPAAHRP SEQ ID 7 P33_8 RWSRAVLFPAAHRP SEQ ID 14 P33_9HRWSRAVLFPAAHRP SEQ ID 15 P33_10 WSRAVLFPAAHRP SEQ ID 16 P33_5AVLFPAAHRP SEQ ID 27Formula III includes compounds falling within the following formula:

-   C¹-C²-C³-C⁴-C⁵-C⁶-C⁷-C⁸-C⁹-C¹⁰-C¹¹-C¹²-C¹³-C¹⁴-C¹⁵-C¹⁶-C¹⁷-C¹⁸-C¹⁹-C²⁰-C²¹-C²²-C²³-C²⁴-C²⁵-C²⁶-C²⁷-C²⁸    or a pharmaceutically acceptable salt thereof, wherein    -   C¹ is absent or G or a small non-naturally occurring amino acid;    -   C² is absent or I or a hydrophobic non-naturally occurring amino        acid;    -   C³ is absent or G or a small non-naturally occurring amino acid;        ;    -   C⁴ is C or S or a polar non-naturally occurring amino acid;    -   C⁵ is V or a hydrophobic non-naturally occurring amino acid;    -   C⁶ is W or a hydrophobic non-naturally occurring amino acid    -   C⁷ is H or a basic non-naturally occurring amino acid;    -   C⁸ is W or a hydrophobic non-naturally occurring amino acid;    -   C⁹ is K or a basic non-naturally occurring amino acid;    -   C¹⁰ is H or a basic non-naturally occurring amino acid;    -   C¹¹ is R or a basic non-naturally occurring amino acid;    -   C¹² is V or a hydrophobic non-naturally occurring amino acid;    -   C¹³ is A or a hydrophobic non-naturally occurring amino acid;    -   C¹⁴ is T or a polar non-naturally occurring amino acid;    -   C¹⁵ is R or a basic non-naturally occurring amino acid;    -   C¹⁶ is F or a hydrophobic non-naturally occurring amino acid;    -   C¹⁷ is T or a polar non-naturally occurring amino acid;    -   C¹⁸ is L or a hydrophobic non-naturally occurring amino acid;    -   C¹⁹ is P;    -   C²⁰ is R or basic non-naturally occurring amino acid;    -   C²¹ is F or a polar non-naturally occurring amino acid;    -   C²² is L or a hydrophobic non-naturally occurring amino acid;    -   C²³ is Q or a polar non-naturally occurring amino acid;    -   C²⁴ is absent or R or a basic non-naturally occurring amino        acid;    -   C²⁵ is absent or R or a basic non-naturally occurring amino        acid;    -   C²⁶ is absent or S or a polar non-naturally occurring amino        acid;    -   C²⁷ is absent or S or a polar non-naturally occurring amino        acid; or    -   C²⁸ is absent or R or a basic non-naturally occurring amino        acid.

Examples of peptides that include some or all of a sequence withinFormula III are shown in Table 3 below.

A peptide consisting of the amino acid sequence ofGIGSVWHWKHRVATRFTLPRFLQ (SEQ ID NO:8) is also referred to herein asPeptide 60_S.

TABLE 3 P60-type peptides falling within Formula III. Name SequenceSEQ ID P60_S GIGSVWHWKHRVATRFTLPRFLQ SEQ ID 8 P60GIGCVWHWKHRVATRFTLPRFLQ SEQ ID 39 P60 derivativesGIGCVWHWKHRVATRFTLPRFLQRR SEQ ID 40 P60 derivativesGIGCVWHWKHRVATRFTLPRFLQRRSS SEQ ID 41 P60 derivativesGIGCVWHWKHRVATRFTLPRFLQRRSSR SEQ ID 42 P60 derivativesIGCVWHWKHRVATRFTLPRFLQ SEQ ID 43 P60 derivativesIGCVWHWKHRVATRFTLPRFLQRR SEQ ID 44 P60 derivativesIGCVWHWKHRVATRFTLPRFLQRRSS SEQ ID 45 P60 derivativesIGCVWHWKHRVATRFTLPRFLQRRSSR SEQ ID 46 P60 derivativesCVWHWKHRVATRFTLPRFLQ SEQ ID 47 P60 derivatives CVWHWKHRVATRFTLPRFLQRRSEQ ID 48 P60 derivatives CVWHWKHRVATRFTLPRFLQRRSS SEQ ID 49P60 derivatives CVWHWKHRVATRFTLPRFLQRRSSR SEQ ID 50

Formula IV includes compounds falling within the following formula:

-   D¹-D²-D³-D⁴-D⁵-D⁶-D⁷-D⁸-D⁹-D¹⁰-D¹¹-D¹²-D¹³-D¹⁴-D¹⁵-D¹⁶-D¹⁷-D¹⁸-D¹⁹-D²⁰-D²¹-D²²-D²³-D²⁴-D²⁵-D²⁶-D²⁷-D²⁸-D²⁹-D³⁰-D³¹-D³²-D³³-D³⁴-D³⁵-D³⁶-D³⁷-D³⁸-D³⁹-D⁴⁰-D⁴¹-D⁴²-D⁴³-D⁴⁴D⁴⁵    -   or a pharmaceutically acceptable salt thereof, wherein    -   D¹ is A or a small non-naturally occurring amino acid;    -   D² is A or a small non-naturally occurring amino acid;    -   D³ is Q or a polar non-naturally occurring amino acid;    -   D⁴ is A or a hydrophobic non-naturally occurring amino acid;    -   D⁵ is T or a polar non-naturally occurring amino acid;    -   D⁶ is G or a small non-naturally occurring amino acid;    -   D⁷ is P;    -   D⁸ is L or a hydrophobic non-naturally occurring amino acid;    -   D⁹ is Q or a polar non-naturally occurring amino acid;    -   D¹⁰ is D or a polar non-naturally occurring amino acid;    -   D¹¹ is N or a polar non-naturally occurring amino acid;    -   D¹² is E or a non-naturally occurring amino acid;    -   D¹³ is L or a hydrophobic non-naturally occurring amino acid;    -   D¹⁴ is P;    -   D¹⁵ is G or a small non-naturally occurring amino acid;    -   D¹⁶ is L, or a hydrophobic non-naturally occurring amino acid;    -   D¹⁷ is D or a polar non-naturally occurring amino acid;    -   D¹⁸ is E or a non-naturally occurring amino acid;    -   D¹⁹ is R or a basic non-naturally occurring amino acid;    -   D²⁰ is P;    -   D²¹ is P;    -   D²² is R or a basic non-naturally occurring amino acid;    -   D²³ is A or a small non-naturally occurring amino acid;    -   D²⁴ is H or a basic non-naturally occurring amino acid;    -   D²⁵ is A or a small non-naturally occurring amino acid;    -   D²⁶ is Q or a polar non-naturally occurring amino acid;    -   D²⁷ is H or a basic non-naturally occurring amino acid;    -   D²⁸ is F or a hydrophobic non-naturally occurring amino acid;    -   D²⁹ is H or a basic non-naturally occurring amino acid;    -   D³⁰ is K or a basic non-naturally occurring amino acid;    -   D³¹ is H or a basic non-naturally occurring amino acid;    -   D³² is Q or a polar non-naturally occurring amino acid;    -   D³³ is L or a hydrophobic non-naturally occurring amino acid;    -   D³⁴ is W or a hydrophobic non-naturally occurring amino acid;    -   D³⁵ is P;    -   D³⁶ is S or a polar non-naturally occurring amino acid;    -   D³⁷ is P;    -   D³⁸ is F or a hydrophobic non-naturally occurring amino acid;    -   D³⁹ is R or a basic non-naturally occurring amino acid;    -   D⁴⁰ is A or a hydrophobic non-naturally occurring amino acid;    -   D⁴¹ is L or a hydrophobic non-naturally occurring amino acid;    -   D⁴² is K or a basic non-naturally occurring amino acid;    -   D⁴³ is P;    -   D⁴⁴ is R or a hydrophobic non-naturally occurring amino acid;    -   D⁴⁵ is P.

In some embodiments, a peptide of Formula IV includes the amino acidsequence as listed in Table 4 below.

A peptide consisting of the amino acid sequenceAAQATGPLQDNELPGLDERPPRAHAQHFHKHQLWPSPFRALKPRP (SEQ ID NO: 5) is alsoreferred to herein is as Peptide 94.

TABLE 4 P94-type peptides falling within Formula IV. Name SequenceSEQ ID P94 AAQATGPLQDNELPGLDERPPRAHAQHFHKHQLWPSPFRAL SEQ ID 5 KPRPP94/63_27 AAQATGPLQDNELPGLDERPP SEQ ID 32 P94/63_26AAQATGPLQDNELPGLDERPPRAHAQHFH SEQ ID 33 P94/63_21PPRAHAQHFHKHQLWPSPFRALKPRP SEQ ID 34 P94/63_9 HQLWPSPFRALKPRP SEQ ID 35

Compounds of Formula V include the following:

-   E¹-E²-E³-E⁴-E⁵-E⁶-E⁷-E⁸-E⁹-E¹⁰-E¹¹-E¹²-E¹³-E¹⁴-E¹⁵-E¹⁶-E¹⁷-E¹⁸-E¹⁹-E²⁰-E²¹-E²²-E²³,    or a pharmaceutically acceptable salt thereof, wherein    -   E¹ is A or a small non-naturally occurring amino acid;    -   E² is H or a basic non-naturally occurring amino acid;    -   E³ is A or a small non-naturally occurring amino acid;    -   E⁴ is Q or a polar non-naturally occurring amino acid;    -   E⁵ is H or a basic non-naturally occurring amino acid;    -   E⁶ is F or a hydrophobic non-naturally occurring amino acid;    -   E⁷ is H or a basic non-naturally occurring amino acid;    -   E⁸ is K or a basic non-naturally occurring amino acid;    -   E⁹ is H or a basic non-naturally occurring amino acid;    -   E¹⁰ is Q or a polar non-naturally occurring amino acid;    -   E¹¹ is L or a hydrophobic non-naturally occurring amino acid;    -   E¹² is W or a hydrophobic non-naturally occurring amino acid;    -   E¹³ is P;    -   E¹⁴ is S;    -   E¹⁵ is P;    -   E¹⁶ is F or a hydrophobic non-naturally occurring amino acid;    -   E¹⁷ is R or a basic non-naturally occurring amino acid;    -   E¹⁸ is A or a small non-naturally occurring amino acid.    -   E¹⁹ is L or a hydrophobic non-naturally occurring amino acid;    -   E²⁰ is K or a basic non-naturally occurring amino acid;    -   E²¹ is P;    -   E²² is R or a basic non-naturally occurring amino acid;    -   E²³ is P.

Examples of P63-type peptides within Formula V includeAHAQHFHKHQLWPSPFRALKPRP (SEQ ID NO:17 also referred to herein as Peptide63 (P63).

Formula VI includes compounds falling within the following formula:

-   F¹-F²-F³-F⁴-F⁵-F⁶-F⁷-F⁸-F⁹-F¹⁰-F¹¹-F¹²-F¹³-F¹⁴-F¹⁵-F¹⁶-F¹⁷-F¹⁸-F¹⁹-F²⁰-F²¹-F²²-F²³-F²⁴    or a pharmaceutically acceptable salt thereof, wherein    -   F¹ is absent or H or a basic non-naturally occurring amino acid;    -   F² is absent or K or a basic non-naturally occurring amino acid;    -   F³ is absent or R or a basic non-naturally occurring amino acid;    -   F⁴ is T or a polar non-naturally occurring amino acid;    -   F⁵ is I or a hydrophobic non-naturally occurring amino acid;    -   F⁶ is P;    -   F⁷ is M or norleucine (Nle) or another hydrophobic non-naturally        occurring amino acid;    -   F⁸ is F or a hydrophobic non-naturally occurring amino acid;    -   F⁹ is V or a hydrophobic non-naturally occurring amino acid;    -   F¹⁰ is P;    -   F¹¹ is E or a non-naturally occurring amino acid;    -   F¹² is S;    -   F¹³ is T or a polar non-naturally occurring amino acid;    -   F¹⁴ is S;    -   F¹⁵ is K or a basic non-naturally occurring amino acid;    -   F¹⁶ is L or a hydrophobic non-naturally occurring amino acid;    -   F¹⁷ is Q or a polar non-naturally occurring amino acid;    -   F¹⁸ is K or a basic non-naturally occurring amino acid;    -   F¹⁹ is F or a polar non-naturally occurring amino acid;    -   F²⁰ is T or polar non-naturally occurring amino acid;    -   F²¹ is S;    -   F²² is W or a hydrophobic non-naturally occurring amino acid;    -   F²³ is F or a polar non-naturally occurring amino acid;    -   F²⁴ is M or norleucine (Nle) or another hydrophobic        non-naturally occurring amino acid;

Examples of peptides that include some of the sequence within Formula VIare shown in Table 5 below.

A peptide consisting of the amino acid sequence of TIPMFVPESTSKLQKFTSWFM(SEQ ID NO:1) is also referred to herein as Peptide 58.

TABLE 5 P58-type peptides falling within Formula VI: Name SequenceSEQ ID P58 TIPMFVPESTSKLQKFTSWFM-amide SEQ ID 1 P58_4 FTSWFM-amideSEQ ID 2 P58_5 LQKFTSWFM-amide SEQ ID 3 P58_10TIPMFVPESTSTLQKFTSWFM-amide SEQ ID 4 P58 derivativeHKRTIPMFVPESTSKLQKFTSWFM-amide SEQ ID 26 P58_7 TIPMFVPESTSKLQ SEQ ID 36P58_12 TIPMFVPESTSTLQ SEQ ID 37 P58_6 TIPMFVPESTS SEQ ID 38

A peptide within Formula I, II, III, IV, V, or VI can be provided aspart of a longer peptide that includes the specified amino acidsequence. For example, the peptide can be provided on a peptide that isless than 200, 150, 125, 100, 75, 50, 25, 24, 23, 22, 21, 20, 19, 18, or17 amino acids. The invention additionally provides a peptide fragmenthaving fewer than the amino acid sequences recited Formula I, II, III,IV, V, or VI. In preferred embodiments, the peptide fragment retains oneor more of the activities associated with the full-length peptide, e.g.,binding to and/or activation of a GPCR receptor, or activity against acondition described herein.

In some embodiments, a peptide within Formula I, II, or III binds aG-protein coupled receptor (GPCR) protein. For example a peptide canbind a MAS1 gene product for a peptide of Formula I or Formula II or apeptide can bind a Mas-related G-protein coupled receptor member X1(Sensory neuron-specific G-protein coupled receptor 4) for a peptide ofFormula III. A peptide can bind a Mas-related G-protein coupled receptormember X2 for a peptide of Formula

I, III, IV or V or a peptide can bind a FMLP-related receptor I for apeptide of Formula II, III, IV or VI.

In some embodiments, a peptide within Formula I, II III, IV, V, or VIactivates a GPCR protein. Activation of a GPCR protein can be measuredusing methods known in the art.

A peptide within Formula I, II, III, IV, V, or VI can be providedconjugated to a second peptide or polypeptide. Examples of secondpeptides or polypeptides are multiple antigenic peptides (MAP) and asignal sequence. Suitable signal sequences include, e.g.,MPSVRSLLRLLAAAAACGAFA (SEQ ID NO:51), MPSVRSLLRLLAAAAACGA (SEQ IDNO:52); MHWKMLLLLLLYYNAEA (SEQ ID NO:53); MSKSCGNNLAAISVGISLLLLLVVC (SEQID NO:54); MAHVPARTSPGPGPQLLLLLLPLFLLLLRDVAG (SEQ ID NO: 55); andMATASPSVFLLMVNGQVES (SEQ ID NO: 56).

In some embodiments, the second peptide or polypeptide is animmunoglobulin sequence (e.g., an IgG sequence). Immunoreactive ligandsfor use as a targeting moiety in the invention include anantigen-recognizing immunoglobulin (also referred to as “antibody”), orantigen-recognizing fragment thereof, e.g., immunoglobulins that canrecognize a tumor-associated antigen. As used herein, “immunoglobulin”refers to any recognized class or subclass of immunoglobulins such asIgG, IgA, IgM, IgD, or IgE.

Preferred are those immunoglobulins which fall within the IgG class ofimmunoglobulins. The immunoglobulin can be derived from any species.Preferably, however, the immunoglobulin is of human, murine, or rabbitorigin. In addition, the immunoglobulin may be polyclonal or monoclonal,but is preferably monoclonal.

Conjugates of the invention may include an antigen-recognizingimmunoglobulin fragment. Such immunoglobulin fragments may include, forexample, the Fab′, F (ab′)2, Fv or Fab fragments, or otherantigen-recognizing immunoglobulin fragments. Such immunoglobulinfragments can be prepared, for example, by proteolytic enzyme digestion,for example, by pepsin or papain digestion, reductive alkylation, orrecombinant techniques. The materials and methods for preparing suchimmunoglobulin fragments are well-known to those skilled in the art. SeeParham, J. Immunology, 131,2895, 1983; Lamoyi et al., J. ImmunologicalMethods, 56,235, 1983.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an analog or mimetic of a corresponding naturally occurringamino acid, as well as to naturally occurring amino acid polymers. Theterms encompass any peptide (including cyclic peptides) or proteincomprising two or more amino acids joined to each other by peptide bondsor modified peptide bonds. “Polypeptide” refers to both short chains,commonly referred to as peptides, oligopeptides or oligomers, and tolonger chains, generally referred to as proteins.

“Polypeptides” include amino acid sequences modified either by naturalprocesses, or by chemical modification techniques which are well knownin the art. Modifications may occur anywhere in a polypeptide, includingthe peptide backbone, the amino acid side-chains, and the amino orcarboxyl termini. Polypeptides can be modified, e.g., by the addition ofcarbohydrate residues to form glycoproteins. The terms “polypeptide,”“peptide” and “protein” include glycoproteins, as well asnon-glycoproteins.

Peptides within the invention can be produced using methods known in theart, e.g., by purifying the peptide sequence from a naturally occurringprotein or peptide. Purification can be performed along with a cleavageor degradation (either enzymatic or non-enzymatic) to produce thedesired peptide using methods known in the art.

Alternatively, products can be biochemically synthesized using, e.g.,solid phase synthesis, partial solid phase synthesis methods, fragmentcondensation, classical solution synthesis. These methods are preferablyused when the peptide is relatively short (i.e., 10 kDa) and/or when itcannot be produced by recombinant techniques (i.e., not encoded by anucleic acid sequence).

Solid phase polypeptide synthesis procedures are well known in the artand further described by John Morrow Stewart and Janis Dillaha Young,Solid Phase Peptide Syntheses (2nd Ed., Pierce Chemical Company, 1984).

Synthetic polypeptides can be purified by preparative high performanceliquid chromatography [Creighton T. (1983) Proteins, structures andmolecular principles. WH Freeman and Co. N.Y.] and the composition ofwhich can be confirmed via amino acid sequencing.

Polypeptides or peptides can alternatively be synthesized usingrecombinant techniques such as those described by Bitter et al., (1987)Methods in Enzymol. 153:516-544, Studier et al. (1990) Methods inEnzymol. 185:60-89, Brisson et al. (1984) Nature 310:511-514, Takamatsuet al. (1987) EMBO J. 6:307-311, Coruzzi et al. (1984) EMBO J.3:1671-1680 and Brogli et al., (1984) Science 224:838-843, Gurley et al.(1986) Mol. Cell. Biol. 6:559-565 and Weissbach & Weissbach, 1988,Methods for Plant Molecular Biology, Academic Press, NY, Section VIII,pp 421-463.

A peptide within the invention may include one or more modifications.For example, it may be provided phosphorylated (typically at a serine,threonine, or tyrosine residue), pegylated, coupled to a biotin moiety,or include a disulfide bond to another peptide, polypeptide or aminoacid. The peptide may be provided in a cyclic form, e.g., as a cyclicpeptide or as a lactam. Alternatively, or in addition, the peptide maybe provided as a branched peptide.

The peptide may be additionally modified (when linear) at its aminoterminus or carboxy terminus. Examples of amino terminal modificationsinclude, e.g., N-glycated, N-alkylated, N-acetylated or N-acylated aminoacid. A terminal modification can include a pegylation. An example of acarboxy terminal modification is a c-terminal amidated amino acid.

A peptide of the invention may contain amino acids other than the 20gene-encoded amino acids. When amino acids are not designated as eitherD- or L-amino acids, the amino acid is either an L-amino acid or couldbe either a D- or L-amino acid, unless the context requires a particularisomer.

The notations used herein for the polypeptide amino acid residues arethose abbreviations commonly used in the art. The less commonabbreviations Abu, Cpa, Nle, Pal, Tle, Dip, 4-Fpa, and Nal stand for2-amino-butyric acid, p-chlorophenylalanine, norleucine,3-pyridyl-2-alanine, tert-leucine, 2,2-diphenylalanine,4-fluoro-phenylalanine, and 3-(2-naphthyl)-alanine or3-(1-naphthyl)-alanine, respectively.

One example of a non-naturally occurring amino acid is an omega-aminoacid, e.g., beta-alanine (beta-Ala), or 3 aminopropionic (3-aP). Otherexamples are non-naturally occurring amino acids, e.g., sarcosine (Sar),β-alanine (β-Ala), 2,3 diaminopropionic (2,3-diaP) oralpha-aminisobutyric acid (Aib); omega-acid is beta-alanine (beta-Ala),or 3 aminopropionic (3-aP); a hydrophobic non-naturally occurring aminoacid, such as t-butylalanine (t BuA), t butylglycine (t BuG), Nmethylisoleucine (N MeIle), norleucine (Nle), methylvaline (Mvl),cyclohexylalanine (Cha), phenylglycine (Phg), NaI, β2-thienylalanine(Thi), 2 naphthylalanine (2 Nal), or 1,2,3,4-tetrahydroisoquinoline-3carboxylic acid (Tic); a basic amino acid, such as ornithine (Orn) orhomoarginine (Har); and a neutral/polar non-naturally occurring aminoacid is citrulline (Cit), Acetyl Lys, or methionine sulfoxide (MSO).

Other non-conventional amino acids are listed in Table 6.

TABLE 6 Non-conventional amino acid Code Non-conventional amino acidCode α-aminobutyric acid Abu L-N-methylalanine Nmalaα-amino-α-methylbutyrate Mgabu L-N-methylarginine Nmargaminocyclopropane- Cpro L-N-methylasparagine Nmasn carboxylateL-N-methylaspartic acid Nmasp aminoisobutyric acid AibL-N-methylcysteine Nmcys aminonorbornyl- Norb L-N-methylglutamine Nmgincarboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine ChexaL-N-methylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisolleucineNmile D-alanine Dal L-N-methylleucine Nmleu D-arginine DargL-N-methyllysine Nmlys D-aspartic acid Dasp L-N-methylmethionine NmmetD-cysteine Dcys L-N-methylnorleucine Nmnle D-glutamine DglnL-N-methylnorvaline Nmnva D-glutamic acid Dglu L-N-methylornithine NmornD-histidine Dhis L-N-methylphenylalanine Nmphe D-isoleucine DileL-N-methylproline Nmpro D-leucine Dleu L-N-methylserine Nmser D-lysineDlys L-N-methylthreonine Nmthr D-methionine Dmet L-N-methyltryptophanNmtrp D-ornithine Dorn L-N-methyltyrosine Nmtyr D-phenylalanine DpheL-N-methylvaline Nmval D-proline Dpro L-N-methylethylglycine NmetgD-serine Dser L-N-methyl-t-butylglycine Nmtbug D-threonine DthrL-norleucine Nle D-tryptophan Dtrp L-norvaline Nva D-tyrosine Dtyrα-methyl-aminoisobutyrate Maib D-valine Dval α-methyl-γ-aminobutyrateMgabu D-α-methylalanine Dmala α-methylcyclohexylalanine MchexaD-α-methylarginine Dmarg α-methylcyclopentylalanine McpenD-α-methylasparagine Dmasn α-methyl-α-napthylalanine ManapD-α-methylaspartate Dmasp α-methylpenicillamine Mpen D-α-methylcysteineDmcys N-(4-aminobutyl)glycine Nglu D-α-methylglutamine DmglnN-(2-aminoethyl)glycine Naeg D-α-methylhistidine DmhisN-(3-aminopropyl)glycine Norn D-α-methylisoleucine DmileN-amino-α-methylbutyrate Nmaabu D-α-methylleucine Dmleu α-napthylalanineAnap D-α-methyllysine Dmlys N-benzylglycine Nphe D-α-methylmethionineDmmet N-(2-carbamylethyl)glycine Ngln D-α-methylornithine DmornN-(carbamylmethyl)glycine Nasn D-α-methylphenylalanine DmpheN-(2-carboxyethyl)glycine Nglu D-α-methylproline DmproN-(carboxymethyl)glycine Nasp D-α-methylserine Dmser N-cyclobutylglycineNcbut D-α-methylthreonine Dmthr N-cycloheptylglycine NchepD-α-methyltryptophan Dmtrp N-cyclohexylglycine Nchex D-α-methyltyrosineDmty N-cyclodecylglycine Ncdec D-α-methylvaline DmvalN-cyclododeclglycine Ncdod D-α-methylalnine Dnmala N-cyclooctylglycineNcoct D-α-methylarginine Dnmarg N-cyclopropylglycine NcproD-α-methylasparagine Dnmasn N-cycloundecylglycine NcundD-α-methylasparatate Dnmasp N-(2,2-diphenylethyl)glycine NbhmD-α-methylcysteine Dnmcys N-(3,3-diphenylpropyl)glycine NbheD-N-methylleucine Dnmleu N-(3-indolylyethyl) glycine NhtrpD-N-methyllysine Dnmlys N-methyl-γ-aminobutyrate NmgabuN-methylcyclohexylalanine Nmchexa D-N-methylmethionine DnmmetD-N-methylornithine Dnmorn N-methylcyclopentylalanine NmcpenN-methylglycine Nala D-N-methylphenylalanine DnmpheN-methylaminoisobutyrate Nmaib D-N-methylproline DnmproN-(1-methylpropyl)glycine Nile D-N-methylserine DnmserN-(2-methylpropyl)glycine Nile D-N-methylserine DnmserN-(2-methylpropyl)glycine Nleu D-N-methylthreonine DnmthrD-N-methyltryptophan Dnmtrp N-(1-methylethyl)glycine NvaD-N-methyltyrosine Dnmtyr N-methyla-napthylalanine NmanapD-N-methylvaline Dnmval N-methylpenicillamine Nmpen γ-aminobutyric acidGabu N-(p-hydroxyphenyl)glycine Nhtyr L-t-butylglycine TbugN-(thiomethyl)glycine Ncys L-ethylglycine Etg penicillamine PenL-homophenylalanine Hphe L-α-methylalanine Mala L-α-methylarginine MargL-α-methylasparagine Masn L-α-methylaspartate MaspL-α-methyl-t-butylglycine Mtbug L-α-methylcysteine McysL-methylethylglycine Metg L-α-methylglutamine Mgln L-α-methylglutamateMglu L-α-methylhistidine Mhis L-α-methylhomo phenylalanine MhpheL-α-methylisoleucine Mile N-(2-methylthioethyl)glycine NmetD-N-methylglutamine Dnmgln N-(3-guanidinopropyl)glycine NargD-N-methylglutamate Dnmglu N-(1-hydroxyethyl)glycine NthrD-N-methylhistidine Dnmhis N-(hydroxyethyl)glycine NserD-N-methylisoleucine Dnmile N-(imidazolylethyl)glycine NhisD-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine NhtrpD-N-methyllysine Dnmlys N-methyl-γ-aminobutyrate NmgabuN-methylcyclohexylalanine Nmchexa D-N-methylmethionine DnmmetD-N-methylornithine Dnmorn N-methylcyclopentylalanine NmcpenN-methylglycine Nala D-N-methylphenylalanine DnmpheN-methylaminoisobutyrate Nmaib D-N-methylproline DnmproN-(1-methylpropyl)glycine Nile D-N-methylserine DnmserN-(2-methylpropyl)glycine Nleu D-N-methylthreonine DnmthrD-N-methyltryptophan Dnmtrp N-(1-methylethyl)glycine NvalD-N-methyltyrosine Dnmtyr N-methyla-napthylalanine NmanapD-N-methylvaline Dnmval N-methylpenicillamine Nmpen γ-aminobutyric acidGabu N-(p-hydroxyphenyl)glycine Nhtyr L-t-butylglycine TbugN-(thiomethyl)glycine Ncys L-ethylglycine Etg penicillamine PenL-homophenylalanine Hphe L-α-methylalanine Mala L-α-methylarginine MargL-α-methylasparagine Masn L-α-methylaspartate MaspL-α-methyl-t-butylglycine Mtbug L-α-methylcysteine McysL-methylethylglycine Metg L-α-methylglutamine Mgln L-α-methylglutamateMglu L-α-methylhistidine Mhis L-α-methylhomophenylalanine MhpheL-α-methylisoleucine Mile N-(2-methylthioethyl)glycine NmetL-α-methylleucine Mleu L-α-methyllysine Mlys L-α-methylmethionine MmetL-α-methylnorleucine Mnle L-α-methylnorvaline Mnva L-α-methylornithineMorn L-α-methylphenylalanine Mphe L-α-methylproline MproL-α-methylserine mser L-α-methylthreonine Mthr L-α-methylvaline MtrpL-α-methyltyrosine Mtyr L-α-methylleucine MvalL-N-methylhomophenylalanine Nmhphe nbhm N-(N-(2,2-diphenylethyl)N-(N-(3,3-diphenylpropyl) carbamylmethyl-glycine Nnbhmcarbamylmethyl(1)glycine Nnbhe 1-carboxy-1-(2,2-diphenyl Nmbchylamino)cyclopropaneModificationsFusion Proteins

A fusion protein may be prepared from a peptide according to the presentinvention by fusion with a portion of an immunoglobulin comprising aconstant region of an immunoglobulin. More preferably, the portion ofthe immunoglobulin comprises a heavy chain constant region which isoptionally and more preferably a human heavy chain constant region. Theheavy chain constant region is most preferably an IgG heavy chainconstant region, and optionally and most preferably is an Fc chain, mostpreferably an IgG Fc fragment that comprises CH2 and CH3 domains.Although any IgG subtype may optionally be used, the IgG1 subtype ispreferred. The Fc chain may optionally be a known or “wild type” Fcchain, or alternatively may be mutated. Non-limiting, illustrative,exemplary types of mutations are described in US Patent Application No.20060034852, published on Feb. 16, 2006, hereby incorporated byreference as if fully set forth herein. The term “Fc chain” alsooptionally comprises any type of Fc fragment.

Several of the specific amino acid residues that are important forantibody constant region-mediated activity in the IgG subclass have beenidentified. Inclusion, substitution or exclusion of these specific aminoacids therefore allows for inclusion or exclusion of specificimmunoglobulin constant region-mediated activity. Furthermore, specificchanges may result in aglycosylation for example and/or other desiredchanges to the Fc chain. At least some changes may optionally be made toblock a function of Fc which is considered to be undesirable, such as anundesirable immune system effect, as described in greater detail below.

Non-limiting, illustrative examples of mutations to Fc which may be madeto modulate the activity of the fusion protein include the followingchanges (given with regard to the Fc sequence nomenclature as given byKabat, from Kabat E A et al: Sequences of Proteins of ImmunologicalInterest. US Department of Health and Human Services, NIH, 1991):220C->S; 233-238 ELLGGP->EAEGAP; 265D->A, preferably in combination with434N->A; 297N->A (for example to block N-glycosylation); 318-322EYKCK->AYACA; 330-331AP->SS; or a combination thereof (see for exampleM. Clark, “Chemical Immunol and Antibody Engineering”, pp 1-31 for adescription of these mutations and their effect). The construct for theFc chain which features the above changes optionally and preferablycomprises a combination of the hinge region with the CH2 and CH3domains.

The above mutations may optionally be implemented to enhance desiredproperties or alternatively to block non-desired properties. Forexample, aglycosylation of antibodies was shown to maintain the desiredbinding functionality while blocking depletion of T-cells or triggeringcytokine release, which may optionally be undesired functions (see M.Clark, “Chemical Immunol and Antibody Engineering”, pp 1-31).Substitution of 331 proline for serine may block the ability to activatecomplement, which may optionally be considered an undesired function(see M. Clark, “Chemical Immunol and Antibody Engineering”, pp 1-31).Changing 330 alanine to serine in combination with this change may alsoenhance the desired effect of blocking the ability to activatecomplement.

Residues 235 and 237 were shown to be involved in antibody-dependentcell-mediated cytotoxicity (ADCC), such that changing the block ofresidues from 233-238 as described may also block such activity if ADCCis considered to be an undesirable function.

Residue 220 is normally a cysteine for Fc from IgG1, which is the siteat which the heavy chain forms a covalent linkage with the light chain.Optionally, this residue may be changed to a serine, to avoid any typeof covalent linkage (see M. Clark, “Chemical Immunol and AntibodyEngineering”, pp 1-31).

The above changes to residues 265 and 434 may optionally be implementedto reduce or block binding to the Fc receptor, which may optionallyblock undesired functionality of Fc related to its immune systemfunctions (see “Binding site on Human IgG1 for Fc Receptors”, Shields etal. vol 276, pp 6591-6604, 2001).

The above changes are intended as illustrations only of optional changesand are not meant to be limiting in any way. Furthermore, the aboveexplanation is provided for descriptive purposes only, without wishingto be bound by a single hypothesis.

Addition of Groups

If a peptide according to the present invention is a linear molecule, itis possible to place various functional groups at various points on thelinear molecule which are susceptible to or suitable for chemicalmodification. Functional groups can be added to the termini of linearforms of the peptide. In some embodiments, the functional groups improvethe activity of the peptide with regard to one or more characteristics,including but not limited to, improvement in stability, penetration(through cellular membranes and/or tissue barriers), tissuelocalization, efficacy, decreased clearance, decreased toxicity,improved selectivity, improved resistance to expulsion by cellularpumps, and the like. For convenience sake and without wishing to belimiting, the free N-terminus of one of the sequences contained in thecompositions of the invention will be termed as the N-terminus of thecomposition, and the free C-terminal of the sequence will be consideredas the C-terminus of the composition. Either the C-terminus or theN-terminus of the sequences, or both, can be linked to a carboxylic acidfunctional groups or an amine functional group, respectively.

Non-limiting examples of suitable functional groups are described inGreen and Wuts, “Protecting Groups in Organic Synthesis”, John Wiley andSons, Chapters 5 and 7, 1991, the teachings of which are incorporatedherein by reference. Preferred protecting groups are those thatfacilitate transport of the active ingredient attached thereto into acell, for example, by reducing the hydrophilicity and increasing thelipophilicity of the active ingredient, these being an example for “amoiety for transport across cellular membranes”.

These moieties can optionally and preferably be cleaved in vivo, eitherby hydrolysis or enzymatically, inside the cell. (Ditter et al., J.Pharm. Sci. 57:783 (1968); Ditter et al., J. Pharm. Sci. 57:828 (1968);Ditter et al., J. Pharm. Sci. 58:557 (1969); King et al., Biochemistry26:2294 (1987); Lindberg et al., Drug Metabolism and Disposition 17:311(1989); and Tunek et al., Biochem. Pharm. 37:3867 (1988), Anderson etal., Arch. Biochem. Biophys. 239:538 (1985) and Singhal et al., FASEB J.1:220 (1987)). Hydroxyl protecting groups include esters, carbonates andcarbamate protecting groups. Amine protecting groups include alkoxy andaryloxy carbonyl groups, as described above for N-terminal protectinggroups. Carboxylic acid protecting groups include aliphatic, benzylicand aryl esters, as described above for C-terminal protecting groups. Inone embodiment, the carboxylic acid group in the side chain of one ormore glutamic acid or aspartic acid residue in a composition of thepresent invention is protected, preferably with a methyl, ethyl, benzylor substituted benzyl ester, more preferably as a benzyl ester.

Non-limiting, illustrative examples of N-terminal protecting groupsinclude acyl groups (—CO—R1) and alkoxy carbonyl or aryloxy carbonylgroups (—CO—O—R1), wherein R1 is an aliphatic, substituted aliphatic,benzyl, substituted benzyl, aromatic or a substituted aromatic group.Specific examples of acyl groups include but are not limited to acetyl,(ethyl)-CO—, n-propyl-CO—, iso-propyl-CO—, n-butyl-CO—, sec-butyl-CO—,t-butyl-CO—, hexyl, lauroyl, palmitoyl, myristoyl, stearyl, oleoylphenyl-CO—, substituted phenyl-CO—, benzyl-CO— and (substitutedbenzyl)-CO—. Examples of alkoxy carbonyl and aryloxy carbonyl groupsinclude CH3-O—CO—, (ethyl)-O—CO—, n-propyl-O—CO—, iso-propyl-O—CO—,n-butyl-O—CO—, sec-butyl-O—CO—, t-butyl-O—CO—, phenyl-O—CO—, substitutedphenyl-O—CO— and benzyl-O—CO—, (substituted benzyl)-O—CO—, Adamantan,naphtalen, myristoleyl, toluen, biphenyl, cinnamoyl, nitrobenzoy,toluoyl, furoyl, benzoyl, cyclohexane, norbornane, or Z-caproic. Inorder to facilitate the N-acylation, one to four glycine residues can bepresent in the N-terminus of the molecule.

The carboxyl group at the C-terminus of the compound can be protected,for example, by a group including but not limited to an amide (i.e., thehydroxyl group at the C-terminus is replaced with —NH₂, —NHR₂ and—NR₂R₃) or ester (i.e. the hydroxyl group at the C-terminus is replacedwith —OR₂). R₂ and R₃ are optionally independently an aliphatic,substituted aliphatic, benzyl, substituted benzyl, aryl or a substitutedaryl group. In addition, taken together with the nitrogen atom, R₂ andR₃ can optionally form a C4 to C8 heterocyclic ring with from about 0-2additional heteroatoms such as nitrogen, oxygen or sulfur. Non-limitingsuitable examples of suitable heterocyclic rings include piperidinyl,pyrrolidinyl, morpholino, thiomorpholino or piperazinyl. Examples ofC-terminal protecting groups include but are not limited to —NH₂,—NHCH₃, —N(CH₃)₂, —NH(ethyl), —N(ethyl)₂, —N(methyl) (ethyl),—NH(benzyl), —N(C1-C4 alkyl)(benzyl), —NH(phenyl), —N(C1-C4 alkyl)(phenyl), —OCH₃, —O-(ethyl), —O-(n-propyl), —O-(n-butyl),—O-(iso-propyl), —O-(sec-butyl), —O-(t-butyl), —O-benzyl and —O-phenyl.

Substitution by Peptidomimetic Moieties

A “peptidomimetic organic moiety” can optionally be substituted foramino acid residues in the composition of this invention both asconservative and as non-conservative substitutions. These moieties arealso termed “non-natural amino acids” and may optionally replace aminoacid residues, amino acids or act as spacer groups within the peptidesin lieu of deleted amino acids. The peptidomimetic organic moietiesoptionally and preferably have steric, electronic or configurationalproperties similar to the replaced amino acid and such peptidomimeticsare used to replace amino acids in the essential positions, and areconsidered conservative substitutions. However such similarities are notnecessarily required. According to preferred embodiments of the presentinvention, one or more peptidomimetics are selected such that thecomposition at least substantially retains its physiological activity ascompared to the native peptide protein according to the presentinvention.

Peptidomimetics may optionally be used to inhibit degradation of thepeptides by enzymatic or other degradative processes. Thepeptidomimetics can optionally and preferably be produced by organicsynthetic techniques. Non-limiting examples of suitable peptidomimeticsinclude D amino acids of the corresponding L amino acids, tetrazol(Zabrocki et al., J. Am. Chem. Soc. 110:5875-5880 (1988)); isosteres ofamide bonds (Jones et al., Tetrahedron Lett. 29: 3853-3856 (1988));LL-3-amino-2-propenidone-6-carboxylic acid (LL-Acp) (Kemp et al., J.Org. Chem. 50:5834-5838 (1985)). Similar analogs are shown in Kemp etal., Tetrahedron Lett. 29:5081-5082 (1988) as well as Kemp et al.,Tetrahedron Lett. 29:5057-5060 (1988), Kemp et al., Tetrahedron Lett.29:4935-4938 (1988) and Kemp et al., J. Org. Chem. 54:109-115 (1987).Other suitable but exemplary peptidomimetics are shown in Nagai andSato, Tetrahedron Lett. 26:647-650 (1985); Di Maio et al., J. Chem. Soc.Perkin Trans., 1687 (1985); Kahn et al., Tetrahedron Lett. 30:2317(1989); Olson et al., J. Am. Chem. Soc. 112:323-333 (1990); Garvey etal., J. Org. Chem. 56:436 (1990). Further suitable exemplarypeptidomimetics includehydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (Miyake et al., J.Takeda Res. Labs 43:53-76 (1989));1,2,3,4-tetrahydro-isoquinoline-3-carboxylate (Kazmierski et al., J. Am.Chem. Soc. 133:2275-2283 (1991)); histidine isoquinolone carboxylic acid(HIC) (Zechel et al., Int. J. Pep. Protein Res. 43 (1991));(2S,3S)-methyl-phenylalanine, (2S,3R)-methyl-phenylalanine,(2R,3S)-methyl-phenylalanine and (2R,3R)-methyl-phenylalanine(Kazmierski and Hruby, Tetrahedron Lett. (1991)).

Exemplary, illustrative but non-limiting non-natural amino acids includebeta-amino acids (beta3 and beta2), homo-amino acids, cyclic aminoacids, aromatic amino acids, Pro and Pyr derivatives, 3-substitutedAlanine derivatives, Glycine derivatives, ring-substituted Phe and TyrDerivatives, linear core amino acids or diamino acids. They areavailable from a variety of suppliers, such as Sigma-Aldrich (USA) forexample.

Chemical Modifications

In the present invention any part of a peptide may optionally bechemically modified, i.e. changed by addition of functional groups. Forexample the side amino acid residues appearing in the native sequencemay optionally be modified, although as described below alternativelyother part(s) of the protein may optionally be modified, in addition toor in place of the side amino acid residues. The modification mayoptionally be performed during synthesis of the molecule if a chemicalsynthetic process is followed, for example by adding a chemicallymodified amino acid. However, chemical modification of an amino acidwhen it is already present in the molecule (“in situ” modification) isalso possible.

The amino acid of any of the sequence regions of the molecule canoptionally be modified according to any one of the following exemplarytypes of modification (in the peptide conceptually viewed as “chemicallymodified”). Non-limiting exemplary types of modification includecarboxymethylation, acylation, phosphorylation, glycosylation or fattyacylation. Ether bonds can optionally be used to join the serine orthreonine hydroxyl to the hydroxyl of a sugar. Amide bonds canoptionally be used to join the glutamate or aspartate carboxyl groups toan amino group on a sugar (Garg and Jeanloz, Advances in CarbohydrateChemistry and Biochemistry, Vol. 43, Academic Press (1985); Kunz, Ang.Chem. Int. Ed. English 26:294-308 (1987)). Acetal and ketal bonds canalso optionally be formed between amino acids and carbohydrates. Fattyacid acyl derivatives can optionally be made, for example, by acylationof a free amino group (e.g., lysine) (Toth et al., Peptides: Chemistry,Structure and Biology, Rivier and Marshal, eds., ESCOM Publ., Leiden,1078-1079 (1990)).

As used herein the term “chemical modification”, when referring to aprotein or peptide according to the present invention, refers to aprotein or peptide where at least one of its amino acid residues ismodified either by natural processes, such as processing or otherpost-translational modifications, or by chemical modification techniqueswhich are well known in the art. Examples of the numerous knownmodifications typically include, but are not limited to: acetylation,acylation, amidation, ADP-ribosylation, glycosylation, GPI anchorformation, covalent attachment of a lipid or lipid derivative,methylation, myristylation, pegylation, prenylation, phosphorylation,ubiquitination, or any similar process.

Other types of modifications optionally include the addition of acycloalkane moiety to a biological molecule, such as a protein, asdescribed in PCT Application No. WO 2006/050262, hereby incorporated byreference as if fully set forth herein. These moieties are designed foruse with biomolecules and may optionally be used to impart variousproperties to proteins.

Furthermore, optionally any point on a protein may be modified. Forexample, pegylation of a glycosylation moiety on a protein mayoptionally be performed, as described in PCT Application No. WO2006/050247, hereby incorporated by reference as if fully set forthherein. One or more polyethylene glycol (PEG) groups may optionally beadded to O-linked and/or N-linked glycosylation. The PEG group mayoptionally be branched or linear. Optionally any type of water-solublepolymer may be attached to a glycosylation site on a protein through aglycosyl linker.

Covalent modifications of the peptides of the present invention areincluded within the scope of this invention. Other types of covalentmodifications of the peptides are introduced into the molecule byreacting targeted amino acid residues with an organic derivatizing agentthat is capable of reacting with selected side chains or the N- orC-terminal residues.

Cysteinyl residues most commonly are reacted with α-haloacetates (andcorresponding amines), such as chloroacetic acid or chloroacetamide, togive carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residuesalso are derivatized by reaction with bromotrifluoroacetone,α-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylpyrocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide also is useful; the reaction ispreferably performed in 0.1M sodium cacodylate at pH 6.0.

Lysinyl and amino-terminal residues are reacted with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing α-amino-containing residues includeimidoesters such as methyl picolinimidate, pyridoxal phosphate,pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid,O-methylisourea, 2,4-pentanedione, and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pKa of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

The specific modification of tyrosyl residues may be made, withparticular interest in introducing spectral labels into tyrosyl residuesby reaction with aromatic diazonium compounds or tetranitromethane. Mostcommonly, N-acetylimidizole and tetranitromethane are used to formO-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosylresidues are iodinated using 125 I or 131 I to prepare labeled proteinsfor use in radioimmunoassay, the chloramine T method described abovebeing suitable.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R—N═C═N—R′), where R and R′ are differentalkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimideor 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. Furthermore,aspartyl and glutamyl residues are converted to asparaginyl andglutaminyl residues by reaction with ammonium ions.

Derivatization with bifunctional agents is useful for crosslinking CHFto a water-insoluble support matrix or surface for use in the method forpurifying anti-CHF antibodies, and vice-versa. Commonly usedcrosslinking agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane,glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with4-azidosalicylic acid, homobifunctional imidoesters, includingdisuccinimidyl esters such as 3,3′-dithiobis(succinimidylpropionate),and bifunctional maleimides such as bis-N-maleimido-1,8-octane.Derivatizing agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatableintermediates that are capable of forming crosslinks in the presence oflight. Alternatively, reactive water-insoluble matrices such as cyanogenbromide-activated carbohydrates and the reactive substrates described inU.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537;and 4,330,440 are employed for protein immobilization.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues, respectively. Theseresidues are deamidated under neutral or basic conditions. Thedeamidated form of these residues falls within the scope of thisinvention.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the α-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MolecularProperties, W. H. Freeman & Co., San Francisco, pp. 79-86 [1983]),acetylation of the N-terminal amine, and amidation of any C-terminalcarboxyl group.

Altered Glycosylation

Peptides of the invention may be modified to have an alteredglycosylation pattern (i.e., altered from the original or nativeglycosylation pattern). As used herein, “altered” means having one ormore carbohydrate moieties deleted, and/or having at least oneglycosylation site added to the original protein.

Glycosylation of proteins is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequences,asparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to peptides of the invention isconveniently accomplished by altering the amino acid sequence of theprotein such that it contains one or more of the above-describedtripeptide sequences (for N-linked glycosylation sites). The alterationmay also be made by the addition of, or substitution by, one or moreserine or threonine residues in the sequence of the original protein(for O-linked glycosylation sites). The protein's amino acid sequencemay also be altered by introducing changes at the DNA level.

Another means of increasing the number of carbohydrate moieties onproteins is by chemical or enzymatic coupling of glycosides to the aminoacid residues of the protein. Depending on the coupling mode used, thesugars may be attached to (a) arginine and histidine, (b) free carboxylgroups, (c) free sulihydryl groups such as those of cysteine, (d) freehydroxyl groups such as those of serine, threonine, or hydroxyproline,(e) aromatic residues such as those of phenylalanine, tyrosine, ortryptophan, or (f) the amide group of glutamine. These methods aredescribed in WO 87/05330, and in Aplin and Wriston, CRC Crit. Rev.Biochem., 22: 259-306 (1981).

Removal of any carbohydrate moieties present on peptides of theinvention may be accomplished chemically or enzymatically. Chemicaldeglycosylation requires exposure of the protein totrifluoromethanesulfonic acid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), leaving the amino acidsequence intact.

Chemical deglycosylation is described by Hakimuddin et al., Arch.Biochem. Biophys., 259: 52 (1987); and Edge et al., Anal. Biochem., 118:131 (1981). Enzymatic cleavage of carbohydrate moieties on proteins canbe achieved by the use of a variety of endo- and exo-glycosidases asdescribed by Thotakura et al., Meth. Enzymol., 138: 350 (1987).

By “variant” is meant a polypeptide that differs from a referencepolypeptide, but retains essential properties. Generally, differencesare limited so that the sequences of the reference polypeptide and thevariant are closely similar overall and, in many regions, identical. Avariant and reference polypeptide may differ in amino acid sequence byone or more substitutions, additions, and/or deletions, in anycombination. A substituted or inserted amino acid residue may or may notbe one encoded by the genetic code. A variant of a polypeptide may be anaturally occurring such as an allelic variant, or it may be a variantthat is not known to occur naturally. Non-naturally occurring variantsof polypeptides may be made by mutagenesis techniques or by directsynthesis.

Generally, the variant differs from the reference polypeptide byconservative amino acid substitutions, whereby a residue is substitutedby another with like characteristics (e.g. acidic, basic, aromatic,etc.). Typical substitutions are among Ala, Val, Leu and Ile; among Serand Thr; among the acidic residues Asp and Glu; among Asn and Gln; andamong the basic residues Lys and Arg; or aromatic residues Phe and Tyr.

Antibodies to Bioactive Peptides

The invention also includes an antibody to a bioactive peptide disclosedherein, or a fragment of the bioactive peptide. In some embodiments, thebioactive peptide is a GPCR ligand.

“Antibody” refers to a polypeptide ligand substantially encoded by animmunoglobulin gene or immunoglobulin genes, or fragments thereof, whichspecifically binds and recognizes an epitope (e.g., an antigen). Theantibody can be provided as, e.g., an intact immunoglobulin or asfragment, e.g., a fragment produced by digestion with variouspeptidases. This includes, e.g., Fab′ and F(ab)′₂ Fv (defined as agenetically engineered fragment containing the variable region of thelight chain and the variable region of the heavy chain expressed as twochains); and (5) Single chain antibody (“SCA”), a genetically engineeredmolecule containing the variable region of the light chain and thevariable region of the heavy chain, linked by a suitable polypeptidelinker as a genetically fused single chain molecule. The term“antibody,” as used herein, also includes antibody fragments eitherproduced by the modification of whole antibodies or those synthesized denovo using recombinant DNA methodologies. It includes polyclonalantibodies, monoclonal antibodies, chimeric antibodies, humanizedantibodies, or single chain antibodies. “Fc” portion of an antibodyrefers to that portion of an immunoglobulin heavy chain that comprisesone or more heavy chain constant region domains, CH1, CH2 and CH3, butdoes not include the heavy chain variable region.

Antibodies are raised against, e.g., an epitope in a peptide of FormulaI, II III, IV, V, or VI. In some embodiments, anti-GPCR peptide ligandantibodies are raised against

-   FLGYCIYLNRKRRGDPAFKRRLRD monomer or dimer (SEQ ID NO. 9)-   FLGYSIYLNRKRRGDPAFKRRLRD (SEQ ID NO. 10)-   IYLNRKRRGDPAFKRRLRD (SEQ ID NO. 11)-   FAFLGYSIYLNRKRRGDPAF (SEQ ID NO. 12)-   FAFLGYCIYLNRKRRGDPAF monomer or dimer (SEQ ID NO. 13)-   FAFLGYSIYLN (SEQ ID NO. 18)-   FAFLGYCIYLN monomer or dimer (SEQ ID NO. 19)-   FAFLGYCIYLNRKRRGDPAFKRRLRD (SEQ ID NO. 20)-   FLGYCIYLN (SEQ ID NO. 21)-   FLGYCIYLNR (SEQ ID NO. 22)-   FLGYCIYLNRKRRGDPAF (SEQ ID NO. 23)-   RGDPAF (SEQ ID NO. 24)-   RRGDPAF (SEQ ID NO. 25)-   GDPAFKRRLRD (SEQ ID NO. 28)-   GDPAF (SEQ ID NO. 29)-   IYLN (SEQ ID NO. 30)-   IYLNRKRRGDPAF (SEQ ID NO. 31)-   SMCHRWSRAVLFPAAHRP- monomer or dimer (SEQ ID NO. 6)-   SMVHRWSRAVLFPAAHRP (SEQ ID NO. 7)-   RWSRAVLFPAAHRP (SEQ ID NO. 14)-   HRWSRAVLFPAAHRP (SEQ ID NO. 15)-   WSRAVLFPAAHRP (SEQ ID NO. 16)-   AVLFPAAHRP (SEQ ID NO. 27)-   GIGSVWHWKHRVATRFTLPRFLQ (SEQ ID NO. 8)-   GIGCVWHWKHRVATRFTLPRFLQ (SEQ ID NO. 39)-   GIGCVWHWKHRVATRFTLPRFLQRR (SEQ ID NO. 40)-   GIGCVWHWKHRVATRFTLPRFLQRRSS (SEQ ID NO. 41)-   GIGCVWHWKHRVATRFTLPRFLQRRSSR (SEQ ID NO. 42)-   IGCVWHWKHRVATRFTLPRFLQ (SEQ ID NO. 43)-   IGCVWHWKHRVATRFTLPRFLQRR (SEQ ID NO. 44)-   IGCVWHWKHRVATRFTLPRFLQRRSS (SEQ ID NO. 45)-   IGCVWHWKHRVATRFTLPRFLQRRSSR (SEQ ID NO. 46)-   CVWHWKHRVATRFTLPRFLQ (SEQ ID NO. 47)-   CVWHWKHRVATRFTLPRFLQRR (SEQ ID NO. 48)-   CVWHWKHRVATRFTLPRFLQRRSS (SEQ ID NO. 49)-   CVWHWKHRVATRFTLPRFLQRRSSR (SEQ ID NO. 50)-   AAQATGPLQDNELPGLDERPPRAHAQHFHKHQLWPSPFRALKPRP (SEQ ID NO. 5)-   AAQATGPLQDNELPGLDERPP (SEQ ID 32)-   AAQATGPLQDNELPGLDERPPRAHAQHFH (SEQ ID NO. 33)-   PPRAHAQHFHKHQLWPSPFRALKPRP (SEQ ID NO. 34)-   HQLWPSPFRALKPRP (SEQ ID NO. 35)-   AHAQHFHKHQLWPSPFRALKPRP (SEQ ID NO. 17)-   TIPMFVPESTSKLQKFTSWFM (SEQ ID NO. 1)-   FTSWFM (SEQ ID NO. 2)-   LQKFTSWFM (SEQ ID NO. 3)-   TIPMFVPESTSTLQKFTSWFM (SEQ ID NO. 4)-   HKRTIPMFVPESTSKLQKFTSWFM (SEQ ID NO. 26)-   TIPMFVPESTSKLQ (SEQ ID NO. 36)-   TIPMFVPESTSTLQ (SEQ ID NO. 37)-   TIPMFVPESTS (SEQ ID NO. 38)

Methods of producing polyclonal and monoclonal antibodies as well asfragments thereof are well known in the art (See for example, Harlow andLane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,New York, 1988, incorporated herein by reference).

Antibody fragments can be prepared by proteolytic hydrolysis of theantibody or by expression in E. coli or mammalian cells (e.g. Chinesehamster ovary cell culture or other protein expression systems) of DNAencoding the fragment. Antibody fragments can be obtained by pepsin orpapain digestion of whole antibodies by conventional methods.

The bioactive peptide antibody can additionally be provided as a peptidecoding corresponding a single complementarity-determining region (CDR).CDR peptides (“minimal recognition units”) can be obtained byconstructing genes encoding the CDR of an antibody of interest. Suchgenes are prepared, for example, by using the polymerase chain reactionto synthesize the variable region from RNA of antibody-producing cells.See, for example, Larrick and Fry [Methods, 2: 106-10 (1991)].

Humanized forms of non-human (e.g., murine) antibodies are chimericmolecules of immunoglobulins, immunoglobulin chains or fragments thereof(such as Fv, Fab, Fab′, F(ab′) or other antigen-binding subsequences ofantibodies) which contain minimal sequence derived from non-humanimmunoglobulin. Humanized antibodies include human immunoglobulins(recipient antibody) in which residues from a complementary determiningregion (CDR) of the recipient are replaced by residues from a CDR of anon-human species (donor antibody) such as mouse, rat or rabbit havingthe desired specificity, affinity and capacity. In some instances, Fvframework residues of the human immunoglobulin are replaced bycorresponding non-human residues. Humanized antibodies may also compriseresidues which are found neither in the recipient antibody nor in theimported CDR or framework sequences. In general, the humanized antibodywill comprise substantially all of at least one, and typically two,variable domains, in which all or substantially all of the CDR regionscorrespond to those of a non-human immunoglobulin and all orsubstantially all of the FR regions are those of a human immunoglobulinconsensus sequence. The humanized antibody optimally also will compriseat least a portion of an immunoglobulin constant region (Fc), typicallythat of a human immunoglobulin [Jones et al., Nature, 321:522-525(1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr.Op. Struct. Biol., 2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as import residues, which aretypically taken from an import variable domain. Humanization can beessentially performed following the method of Winter and co-workers[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such humanized antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries [Hoogenboom and Winter, J.Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581(1991)]. The techniques of Cole et al. and Boerner et al. are alsoavailable for the preparation of human monoclonal antibodies (Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)]. Similarly,human antibodies can be made by introduction of human immunoglobulinloci into transgenic animals, e.g., mice in which the endogenousimmunoglobulin genes have been partially or completely inactivated. Uponchallenge, human antibody production is observed, which closelyresembles that seen in humans in all respects, including generearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the followingscientific publications: Marks et al., Bio/Technology 10,: 779-783(1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996);Neuberger, Nature Biotechnology 14: 826 (1996); and Lonberg and Huszar,Intern. Rev. Immunol. 13, 65-93 (1995).

The antibody preferably binds specifically (or selectively) to a GPCRpeptide ligand. The phrase “specifically (or selectively) binds” to anantibody or “specifically (or selectively) immunoreactive with,” whenreferring to a protein or peptide, refers to a binding reaction that isdeterminative of the presence of the protein in a heterogeneouspopulation of proteins and other biologics. Thus, under designatedimmunoassay conditions, the specified antibodies bind to a particularprotein at least two times the background and do not substantially bindin a significant amount to other proteins present in the sample.Specific binding to an antibody under such conditions may require anantibody that is selected for its specificity for a particular protein.A variety of immunoassay formats may be used to select antibodiesspecifically immunoreactive with a particular protein. For example,solid-phase ELISA immunoassays are routinely used to select antibodiesspecifically immunoreactive with a protein (see, e.g., Harlow & Lane,Antibodies, A Laboratory Manual (1988), for a description of immunoassayformats and conditions that can be used to determine specificimmunoreactivity). Typically a specific or selective reaction will be atleast twice background signal or noise and more typically more than 10to 100 times background.

If desired, the antibody can be provided conjugated or coupled to adetectable label, a radioactive label, an enzyme, a fluorescent label, aluminescent label, a bioluminescent label, or a therapeutic agent.

Methods of Treatment

According to an additional aspect of the present invention there isprovided a method of treating disease, disorder or condition, asdescribed hereinabove, in a subject.

The subject according to the present invention is a mammal, preferably ahuman which is diagnosed with one of the disease, disorder or conditionsdescribed hereinabove, or alternatively is predisposed to at least onetype of disease, disorder or conditions described hereinabove.

As used herein the term “treating” refers to preventing, curing,reversing, attenuating, alleviating, minimizing, suppressing or haltingthe deleterious effects of the above-described diseases, disorders orconditions.

Treating, according to the present invention, can be effected byspecifically upregulating the expression of at least one of thepolypeptides of the present invention in the subject.

Optionally, upregulation may be effected by administering to the subjectat least one of the polypeptides of the present invention (e.g.,recombinant or synthetic) or an active portion thereof, as describedherein. The polypeptide or peptide may optionally be administered in aspart of a pharmaceutical composition, described in more detail below.

It will be appreciated that treatment of the above-described diseasesaccording to the present invention may be combined with other treatmentmethods known in the art (i.e., combination therapy). Thus, treatment ofmalignancies using the agents of the present invention may be combinedwith, for example, radiation therapy, antibody therapy and/orchemotherapy.

Alternatively or additionally, an upregulating method may optionally beeffected by specifically upregulating the amount (optionally expression)in the subject of at least one of the polypeptides of the presentinvention or active portions thereof.

Upregulating expression of the therapeutic peptides of the presentinvention may be effected via the administration of at least one of theexogenous polynucleotide sequences of the present invention, ligatedinto a nucleic acid expression construct designed for expression ofcoding sequences in eukaryotic cells (e.g., mammalian cells).Accordingly, the exogenous polynucleotide sequence may be a DNA or RNAsequence encoding the peptides of the present invention or activeportions thereof.

It will be appreciated that the nucleic acid construct can beadministered to the individual employing any suitable mode ofadministration including in vivo gene therapy (e.g., using viraltransformation as described hereinabove). Alternatively, the nucleicacid construct is introduced into a suitable cell via an appropriategene delivery vehicle/method (transfection, transduction, homologousrecombination, etc.) and an expression system as needed and then themodified cells are expanded in culture and returned to the individual(i.e., ex-vivo gene therapy).

Such cells (i.e., which are transfected with the nucleic acid constructof the present invention) can be any suitable cells, such as kidney,bone marrow, keratinocyte, lymphocyte, adult stem cells, cord bloodcells, embryonic stem cells which are derived from the individual andare transfected ex vivo with an expression vector containing thepolynucleotide designed to express the polypeptide of the presentinvention as described hereinabove.

Administration of the ex vivo transfected cells of the present inventioncan be effected using any suitable route such as intravenous, intraperitoneal, intra kidney, intra gastrointestinal track, subcutaneous,transcutaneous, intramuscular, intracutaneous, intrathecal, epidural andrectal. According to presently preferred embodiments, the ex vivotransfected cells of the present invention are introduced to theindividual using intravenous, intra kidney, intra gastrointestinal trackand/or intra peritoneal administrations.

The ex vivo transfected cells of the present invention can be derivedfrom either autologous sources such as self bone marrow cells or fromallogeneic sources such as bone marrow or other cells derived fromnon-autologous sources. Since non-autologous cells are likely to inducean immune reaction when administered to the body several approaches havebeen developed to reduce the likelihood of rejection of non-autologouscells. These include either suppressing the recipient immune system orencapsulating the non-autologous cells or tissues in immunoisolating,semipermeable membranes before transplantation.

Encapsulation techniques are generally classified as microencapsulation,involving small spherical vehicles and macroencapsulation, involvinglarger flat-sheet and hollow-fiber membranes (Uludag, H. et al.Technology of mammalian cell encapsulation. Adv Drug Deliv Rev. 2000;42: 29-64).

Methods of preparing microcapsules are known in the arts and include forexample those disclosed by Lu MZ, et al., Cell encapsulation withalginate and alpha-phenoxycinnamylidene-acetylated poly(allylamine).Biotechnol Bioeng. 2000, 70: 479-83, Chang T M and Prakash S. Proceduresfor microencapsulation of enzymes, cells and genetically engineeredmicroorganisms. Mol Biotechnol. 2001, 17: 249-60, and Lu MZ, et al., Anovel cell encapsulation method using photosensitive poly(allylaminealpha-cyanocinnamylideneacetate). J Microencapsul. 2000, 17: 245-51.

For example, microcapsules are prepared by complexing modified collagenwith a terpolymer shell of 2-hydroxyethyl methylacrylate (HEMA),methacrylic acid (MAA) and methyl methacrylate (MMA), resulting in acapsule thickness of 2-5 μm. Such microcapsules can be furtherencapsulated with additional 2-5 μm ter-polymer shells in order toimpart a negatively charged smooth surface and to minimize plasmaprotein absorption (Chia, S. M. et al. Multi-layered microcapsules forcell encapsulation Biomaterials. 2002 23: 849-56).

Other microcapsules are based on alginate, a marine polysaccharide(Sambanis, A. Encapsulated islets in diabetes treatment. DiabetesThechnol. Ther. 2003, 5: 665-8) or its derivatives. For example,microcapsules can be prepared by the polyelectrolyte complexationbetween the polyanions sodium alginate and sodium cellulose sulphatewith the polycation poly(methylene-co-guanidine) hydrochloride in thepresence of calcium chloride.

Pharmaceutical Compositions and Delivery Thereof

The bioactive peptide ligand is typically provided in a pharmaceuticallyacceptable carrier suitable for administering the pharmaceuticalcomposition to a human patient. As would be appreciated by one of skillin this art, the carriers may be chosen based on the route ofadministration as described below, the location of the target issue, thedrug being delivered, the time course of delivery of the drug, etc.

The term “pharmaceutically acceptable carrier” means a non-toxic, inertsolid, semi-solid or liquid filler, diluent, encapsulating material orformulation auxiliary of any type. One exemplary pharmaceuticallyacceptable carrier is physiological saline. Other physiologicallyacceptable carriers and their formulations are known to one skilled inthe art and described, for example, in Remington's PharmaceuticalSciences, (18^(th) edition),. A. Gennaro, 1990, Mack Publishing Company,Easton, Pa. Some examples of materials which can serve aspharmaceutically acceptable carriers include, but are not limited to,sugars such as lactose, glucose and sucrose; starches such as cornstarch and potato starch; cellulose and its derivatives such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; powderedtragacanth; malt; gelatin; talc; excipients such as cocoa butter andsuppository waxes; oils such as peanut oil, cottonseed oil; saffloweroil; sesame oil; olive oil; corn oil and soybean oil; glycols such aspropylene glycol; esters such as ethyl oleate and ethyl laurate; agar;detergents such as TWEEN™ 80; buffering agents such as magnesiumhydroxide and aluminum hydroxide; alginic acid; pyrogen-free water;isotonic saline; Ringer's solution; ethyl alcohol; and phosphate buffersolutions, as well as other non-toxic compatible lubricants such assodium lauryl sulfate and magnesium stearate, as well as coloringagents, releasing agents, coating agents, sweetening, flavoring andperfuming agents, preservatives and antioxidants can also be present inthe composition, according to the judgment of the formulator.

By “pharmaceutically acceptable salt” is meant non-toxic acid additionsalts or metal complexes which are commonly used in the pharmaceuticalindustry. Examples of acid addition salts include organic acids such asacetic, lactic, pamoic, maleic, citric, malic, ascorbic, succinic,benzoic, palmitic, suberic, salicylic, tartaric, methanesulfonic,toluenesulfonic, or trifluoroacetic acids or the like; polymeric acidssuch as tannic acid, carboxymethyl cellulose, or the like; and inorganicacids such as hydrochloric acid, hydrobromic acid, sulfuric acidphosphoric acid, or the like. Metal complexes include zinc, iron, andthe like.

The pharmaceutical compositions can be administered to a patient by anymeans known in the art including oral and parenteral routes. The term“patient”, as used herein, refers to humans as well as non-humans,including, for example, mammals, birds, reptiles, amphibians and fish.Preferably, the non-humans are mammals (e.g., a rodent (including amouse or rat), a rabbit, a monkey, a dog, a cat, sheep, cow, pig,horse). The non-human animal could alternatively be a bird, e.g., achicken or turkey.

In certain embodiments parenteral routes are preferred since they avoidcontact with the digestive enzymes that are found in the alimentarycanal. According to such embodiments, inventive compositions including atherapeutic agent may be administered by injection (e.g., intravenous,subcutaneous or intramuscular, intraperitoneal injection), rectally,vaginally, topically (as by powders, creams, ointments, or drops), or byinhalation (as by sprays), intranasal, pulmonary, or intrabuccal.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions, may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension, or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables. Ina particularly preferred embodiment, a therapeutic agent is suspended ina carrier fluid comprising 1% (w/v) sodium carboxymethyl cellulose and0.1% (v/v) TWEEN80™. The injectable formulations can be sterilized, forexample, by filtration through a bacteria-retaining filter, or byincorporating sterilizing agents in the form of sterile solidcompositions which can be dissolved or dispersed in sterile water orother sterile injectable medium prior to use.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the therapeutic agent withsuitable non-irritating excipients or carriers such as cocoa butter,polyethylene glycol, or a suppository wax which are solid at ambienttemperature but liquid at body temperature and therefore melt in therectum or vaginal cavity and release the therapeutic agent.

Dosage forms for topical or transdermal administration of apharmaceutical composition including a therapeutic agent includeointments, pastes, creams, lotions, gels, powders, solutions, sprays,inhalants, or patches. The therapeutic agent is admixed under sterileconditions with a pharmaceutically acceptable carrier and any neededpreservatives or buffers as may be required. Ophthalmic formulations,ear drops and eye drops are also contemplated as being within the scopeof this invention. The ointments, pastes, creams and gels may contain,in addition to the therapeutic agents of this invention, excipients suchas animal and vegetable fats, oils, waxes, paraffins, starch,tragacanth, cellulose derivatives, polyethylene glycols, silicones,bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.Transdermal patches have the added advantage of providing controlleddelivery of a compound to the body. Such dosage forms can be made bydissolving or dispensing the therapeutic agents in a proper medium.Absorption enhancers can also be used to increase the flux of thecompound across the skin. The rate can be controlled by either providinga rate controlling membrane or by dispersing the therapeutic agents in apolymer matrix or gel.

Powders and sprays can also contain excipients such as lactose, talc,silicic acid, aluminum hydroxide, calcium silicates and polyamidepowder, or mixtures of these drugs. Sprays can additionally containcustomary propellants such as chlorofluorohydrocarbons.

When administered orally, the therapeutic agent is optionallyencapsulated. A variety of suitable encapsulation systems are known inthe art (“Microcapsules and Nanoparticles in Medicine and Pharmacy,”Edited by Doubrow, M., CRC Press, Boca Raton, 1992; Mathiowitz andLanger J. Control. Release 5:13, 1987; Mathiowitz et al., ReactivePolymers 6:275, 1987; Mathiowitz et al., J. Appl. Polymer Sci. 35:755,1988; Langer Acc. Chem. Res. 33:94, 2000; Langer J. Control. Release62:7, 1999; Uhrich et al., Chem. Rev. 99:3181, 1999; Zhou et al., J.Control. Release 75:27, 2001; and Hanes et al., Pharm. Biotechnol.6:389, 1995). For example, the therapeutic agent can be encapsulatedwithin biodegradable polymeric microspheres or liposomes. Examples ofnatural and synthetic polymers useful in the preparation ofbiodegradable microspheres include carbohydrates such as alginate,cellulose, polyhydroxyalkanoates, polyamides, polyphosphazenes,polypropylfumarates, polyethers, polyacetals, polycyanoacrylates,biodegradable polyurethanes, polycarbonates, polyanhydrides,polyhydroxyacids, poly(ortho esters) and other biodegradable polyesters.Examples of lipids useful in liposome production include phosphatidylcompounds, such as phosphatidylglycerol, phosphatidylcholine,phosphatidylserine, phosphatidylethanolamine, sphingolipids,cerebrosides and gangliosides.

Pharmaceutical compositions for oral administration can be liquid orsolid. Liquid dosage forms suitable for oral administration of inventivecompositions include pharmaceutically acceptable emulsions,microemulsions, solutions, suspensions, syrups and elixirs. In additionto an encapsulated or unencapsulated therapeutic agent, the liquiddosage forms may contain inert diluents commonly used in the art suchas, for example, water or other solvents, solubilizing agents andemulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate,ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol,1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed,groundnut, corn, germ, olive, castor and sesame oils), glycerol,tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid estersof sorbitan and mixtures thereof. Besides inert diluents, the oralcompositions can also include adjuvants, wetting agents, emulsifying andsuspending agents, sweetening, flavoring and perfuming agents. As usedherein, the term “adjuvant” refers to any compound which is anonspecific modulator of the immune response. In certain preferredembodiments, the adjuvant stimulates the immune response. Any adjuvantmay be used in accordance with the present invention. A large number ofadjuvant compounds are known in the art (Allison, Dev. Biol. Stand.92:3, 1998; Unkeless et al., Annu. Rev. Immunol. 6:251, 1998; andPhillips et al., Vaccine 10: 151, 1992).

Solid dosage forms for oral administration include capsules, tablets,pills, powders and granules. In such solid dosage forms, theencapsulated or unencapsulated therapeutic agent is mixed with at leastone inert, pharmaceutically acceptable excipient or carrier such assodium citrate or dicalcium phosphate and/or (a) fillers or extenderssuch as starches, lactose, sucrose, glucose, mannitol and silicic acid,(b) binders such as, for example, carboxymethylcellulose, alginates,gelatin, polyvinylpyrrolidinone, sucrose and acacia, (c) humectants suchas glycerol, (d) disintegrating agents such as agar-agar, calciumcarbonate, potato or tapioca starch, alginic acid, certain silicates andsodium carbonate, (e) solution retarding agents such as paraffin, (f)absorption accelerators such as quaternary ammonium compounds, (g)wetting agents such as, for example, cetyl alcohol and glycerolmonostearate, (h) absorbents such as kaolin and bentonite clay and (i)lubricants such as talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate and mixtures thereof. In thecase of capsules, tablets and pills, the dosage form may also comprisebuffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like. The solid dosage forms of tablets, dragees, capsules, pillsand granules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart.

The exact dosage of the therapeutic agent is chosen by the individualphysician in view of the patient to be treated. In general, dosage andadministration are adjusted to provide an effective amount of thetherapeutic agent to the patient being treated. As used herein, the“effective amount” of an therapeutic agent refers to the amountnecessary to elicit the desired biological response. As will beappreciated by those of ordinary skill in this art, the effective amountof therapeutic agent may vary depending on such factors as the desiredbiological endpoint, the drug to be delivered, the target tissue, theroute of administration, etc. For example, the effective amount oftherapeutic agent containing an anti-cancer drug might be the amountthat results in a reduction in tumor size by a desired amount over adesired period of time. Additional factors which may be taken intoaccount include the severity of the disease state; age, weight andgender of the patient being treated; diet, time and frequency ofadministration; drug combinations; reaction sensitivities; andtolerance/response to therapy. Long acting pharmaceutical compositionsmight be administered every 3 to 4 days, every week, or once every twoweeks depending on half-life and clearance rate of the particularcomposition.

The therapeutic agents of the invention are preferably formulated indosage unit form for ease of administration and uniformity of dosage.The expression “dosage unit form” as used herein refers to a physicallydiscrete unit of therapeutic agent appropriate for the patient to betreated. It will be understood, however, that the total daily usage ofthe compositions of the present invention will be decided by theattending physician within the scope of sound medical judgment. For anytherapeutic agent, the therapeutically effective dose can be estimatedinitially either in cell culture assays or in animal models, usuallymice, rabbits, dogs, or pigs. The animal model is also used to achieve adesirable concentration range and route of administration. Suchinformation can then be used to determine useful doses and routes foradministration in humans. Therapeutic efficacy and toxicity oftherapeutic agents can be determined by standard pharmaceuticalprocedures in cell cultures or experimental animals, e.g., ED50 (thedose is therapeutically effective in 50% of the population) and LD50(the dose is lethal to 50% of the population). The dose ratio of toxicto therapeutic effects is the therapeutic index and it can be expressedas the ratio, LD50/ED50. Pharmaceutical compositions which exhibit largetherapeutic indices are preferred. The data obtained from cell cultureassays and animal studies is used in formulating a range of dosage forhuman use.

If several different therapeutic modalities (e.g., with differenttherapeutic agents) are to be administered simultaneously then they maybe combined into a single pharmaceutical composition. Alternatively,they may be prepared as separate compositions that are then mixed orsimply administered one after the other. If several differenttherapeutic agents (e.g., with different therapeutic agents) are to beadministered at different times then they are preferably prepared asseparate compositions. If additional drugs are going to be included in acombination therapy they can be added to one or more of thesetherapeutic agents or prepared as separate compositions.

A peptide could be chemically modified in order to alter its propertiessuch as biodistribution, pharmacokinetics and solubility. Variousmethods have been used to increase the solubility and stability ofdrugs, among them the use of organic solvents, their incorporationwithin emulsions or liposomes, the adjustment of pH, their chemicalmodifications and their complexation with the cyclodextrins. Thecyclodextrins are oligosaccharides cyclic family, which include six,seven or eight units of glucopyranose. Due to sterics interactions, thecyclodextrins form a cycle structure in the shape of a cone with aninternal cavity. Those are compounds chemically stable that can bemodified. The cyclodextrins hosts form complexes with varioushydrophobic guests in their cavity. The cyclodextrins are used for thesolubilization and encapsulation of drugs.

Liposomes and Controlled Release:

In order to design a drug delivery system, various kinds of highperformance carrier materials are being developed to deliver thenecessary amount of drug to the targeted site for a necessary period oftime, both efficiently and precisely.

-   Cyclodextrins, biodegradable or non biodegradable polymers,    liposomes, emulsions. Multiple emulsions are potential candidates    for such a role, because of their ability to alter physical,    chemical and biological properties of guest molecules.    There are number of drug delivery systems including but not limited    to polymer microcapsules, microparticles, nanoparticles, liposomes    and emulsion. Many of these are prepared from synthetic    biodegradable polymers such as polyanhydrides and poly hydroxy    acids. In these systems the drugs incorporate in polymeric    microspheres, which release the drug inside the organism in small    and controlled daily doses during days months or until years.

Several polymers already were tested in controlled release systems. Suchas: poiyuretans for its elasticity, polysiloxans or silicons for being agood one insulating, polymethyl-metacrilate for its physical form;polyvinilalcohol for its hydrofobicity and resistance, polyethilene forits hardness and impermeability (Gilding, D. K. Biodegradable polymers.Biocompat. Clin. Impl. Mater. 2:209-232, 1981). Biodegradable polymersand biocompatible polymers, have been extensively investigated asvehicle for controlled release systems due to their ability to undergosurface degradation. These kind of polymers can be chose from:poly(2-hydroxi-ethylmetacrilate), polyacrilamide, polymer from lacticacid (PLA), from glicolic acid (PGA), and the respective onesco-polymers, (PLGA) and the poly(anidrides), as described by Tamada andLanger, J. Biomater. Sci. Polym. Edn, 3(4):315-353.

Suitable controlled release vehicles include, but are not limited to,biocompatible polymers, other polymeric matrices, capsules,microcapsules, nanocapsules, microparticles, nanoparticies, boluspreparations, osmotic pumps, diffusion devices, liposomes, lipospheresand transdermal delivery systems, implantable or not.

Satisfactory systems of controlled release include, but are not limitedto, the ciclodextrines, biocompatible polymers, biodegradable polymers,other polymeric matrixes, capsules, micro-capsules, microparticles,bolus preparations, osmotic pumps, diffusion devices, lipossomes,lipoesferes, and systems of transdermic administration. Othercompositions of controlled release include liquids that, when submittedthe temperature changes, form a solid or a gel in situ.

Liposomes are lipid vesicles that include aqueous internal compartmentsin which molecules, for example drugs, are encapsulated with theobjective of reaching a controlled release of the drug afteradministration in individuals. Many different techniques have beenproposed for the preparation of liposomes [U.S. Pat. No. 4,552,803,Lenk; U.S. Pat. No. 4,310,506, Baldeschwieler; U.S. Pat. No. 4,235,871,Papahadjopoulos; U.S. Pat. No. 4,224,179, Schneider; U.S. Pat. No.4,078,052, Papahadjopoulos; U.S. Pat. No. 4,394,372, Tailor; U.S. Pat.No. 4,308,166, Marchetti; U.S. Pat. No. 4,485,054, Mezei; and U.S. Pat.No. 4,508,703, Redziniak; Woodle and Papahadjopoulos, Methods Enzymol.171:193-215 (1989];. Unilamellar vesicles display a single membrane[Huang, Biochemistry 8:334-352 (1969] while muitilamellar vesicles(MLVs) have numerous concentric membranes [Bangham et al., J. Mol. Biol.13:238-252 (1965]. The procedure of Bangham [J. Mol. Biol. 13:238-252(1965] produces “ordinary MLVs”, that present unequal solutedistributions among the aqueous compartments and, consequently,differences of osmotic pressure. Lenk et al. (U.S. Pat. Nos. 4,522,803;5,030,453 and 5,169,637), Fountain et al. (U.S. Pat. No. 4,588,578),Cullis et al. (U.S. Pat. No. 4,975,282) and Gregoriadis et al. (Pat.W.O. 99/65465) introduced methods for the preparation of MLVs thatpresent substantially equal solute distributions among the compartments.Similar solute distributions among the different compartments mean alarger drug encapsulation efficiency as well as smaller differences ofosmotic pressure that turns these MLVs more stable than ordinary MLVs.Unilamellar vesicles can be produced by sonication of MLVs[Papahadjopoulos et al. (1968)] or by extrusion through polycarbonatemembranes [Cullis et al. (U.S. Pat. No. 5,008,050) and Loughrey et al.(U.S. Pat. No. 5,059,421)].

Satisfactory lipids include for example, phosphatidylcholine,phosphatidylserine, phosphatidylglycerol, cardiolipin, cholesterol,phosphatidic acid, sphingolipids, glycolipids, fatty acids, sterols,phosphatidylethanolamine, polymerizable lipids in their polymerized ornon-polymerized form, mixture of these lipids.

The composition of the liposomes can be manipulated such as to turn themspecific for an organ or a cell type. The targeting of liposomes hasbeen classified either on the basis of anatomical factors or on thebasis of the mechanism of their interaction with the environment. Theanatomical classification is based on their level of selectivity, forexample, organ-specific or cell-specific. From the point of view of themechanisms, the-targeting can be considered as passive or active.

The passive targeting exploits the natural tendency of conventionalliposomes to be captured by the cells of the reticulo-endotheliaisystem, i.e. mainly the fixed macrophages in the liver, spleen and bonemarrow.

Sterically stabilized liposomes (also well-known as “PEG-liposomes”) arecharacterized by a reduced rate of elimination from the bloodcirculation [Lasic and Martin, Stealth Liposomes, CRC Press, Inc., BocaRaton, Fla. (1995)].

PEG-liposomes present a polyethylene glycol polymer conjugated to thehead group of some phospholipid that reduces their interaction withplasma proteins, such as opsonins, and reduces the rate of their uptakeby cells. The resulting steric barrier allows these liposomes to remainfor a longer period of time within the circulation than conventionalliposomes [Lasic and Martin, Stealth Liposomes, CRC Press, Inc., BocaRaton, Fla. (1995); Woodle et al., Biochim.

Biophys. Acta 1105:193-200 (1992); Litzinger et al., Biochim. Biophys.Acta 1190:99-107 (1994); Bedu Addo, et al., Pharm. Res. 13:718-724(1996]. The drug encapsulation within PEG-liposomes has resulted in theimprovement of the effectiveness of many chemotherapeutic agents [Lasicand Martin, Stealth liposomes, CRC Press, Inc., Boca Raton, Fla. (1995)]and bioactive peptides [Allen T. M. In: Liposomes, New Systems, NewTrends in their Applications (F. Puisieux, P. Couvreur, J. Delattre,J.-P. Devissaguet Ed.), Editions de la Sante, France, 1995, pp. 125].

Studies in this area demonstrated that different factors affect theeffectiveness of PEG-liposomes. Ideally, the diameter of the vesiclesshould be below 200 nm, the number of units in PEG of approximately2.000 and the proportion of Pegylated lipid from 3 to 5 mol % [Lasic andMartin, Stealth Liposomes, CRC Press, Inc., Boca Raton, Fla. (1995);Woodle et al., Biochim. Biophys. Acta 1105:193-200 (1992); Litzinger etal., Biochim. Biophys. Acta 1190:99-107 (1994); Bedu Addo et al., Pharm.Res. 13:718-724 (1996)].

The active targeting involves alteration of liposomes through theirassociation with a liguand, such as a monoclonal antibody, a sugar, aglycolipid, protein, a polymer or by changing the lipid composition orthe liposome size to target them to organs and cells different fromthose which accumulate conventional liposomes.

Mucosal Delivery Enhancing Agents

“Mucosal delivery enhancing agents” are defined as chemicals and otherexcipients that, when added to a formulation comprising water, saltsand/or common buffers and peptide within the present invention (thecontrol formulation) produce a formulation that produces a significantincrease in transport of peptide across a mucosa as measured by themaximum blood, serum, or cerebral spinal fluid concentration (Cmax) orby the area under the curve, AUC, in a plot of concentration versustime. A mucosa includes the nasal, oral, intestional, buccal,bronchopulmonary, vaginal, and rectal mucosal surfaces and in factincludes all mucus-secreting membranes lining all body cavities orpassages that communicate with the exterior. Mucosal delivery enhancingagents are sometimes called carriers.

Compositions and Methods of Sustained Release

The present invention provides improved mucosal (e.g., nasal) deliveryof a formulation comprising the peptide within the present invention incombination with one or more mucosal delivery-enhancing agents and anoptional sustained release-enhancing agent or agents. Mucosaldelivery-enhancing agents of the present invention yield an effectiveincrease in delivery, e.g., an increase in the maximal plasmaconcentration (Cmax) to enhance the therapeutic activity ofmucosally-administered peptide. A second factor affecting therapeuticactivity of the peptide in the blood plasma and CNS is residence time(RT). Sustained release-enhancing agents, in combination with intranasaldelivery-enhancing agents, increase Cmaxand increase residence time (RT)of the peptide. Polymeric delivery vehicles and other agents and methodsof the present invention that yield sustained release-enhancingformulations, for example, polyethylene glycol (PEG), are disclosedherein. Within the mucosal delivery formulations and methods of theinvention, the peptide is frequently combined or coordinatelyadministered with a suitable carrier or vehicle for mucosal delivery. Asused herein, the term “carrier” means a pharmaceutically acceptablesolid or liquid filler, diluent or encapsulating material. Awater-containing liquid carrier can contain pharmaceutically acceptableadditives such as acidifying agents, alkalizing agents, antimicrobialpreservatives, antioxidants, buffering agents, chelating agents,complexing agents, solubilizing agents, humectants, solvents, suspendingand/or viscosity-increasing agents, tonicity agents, wetting agents orother biocompatible materials. A tabulation of ingredients listed by theabove categories, can be found in the U.S. Pharmacopeia NationalFormulary, 1857-1859, (1990). As used herein, “mucosaldelivery-enhancing agents” include agents which enhance the release orsolubility (e.g., from a formulation delivery vehicle), diffusion rate,penetration capacity and timing, uptake, residence time, stability,effective half-life, peak or sustained concentration levels, clearanceand other desired mucosal delivery characteristics (e.g., as measured atthe site of delivery, or at a selected target site of activity such asthe bloodstream or central nervous system) of the peptide or otherbiologically active compound(s). Within certain aspects of theinvention, absorption-promoting agents for coordinate administration orcombinatorial formulation with the peptide of the invention are selectedfrom small hydrophilic molecules, including but not limited to, dimethylsulfoxide (DMSO), dimethylformamide, ethanol, propylene glycol, and the2-pyrrolidones. Alternatively, long-chain amphipathic molecules, forexample, deacylmethyl sulfoxide, azone, sodium laurylsulfate, oleicacid, and the bile salts, may be employed to enhance mucosal penetrationof the peptide. In additional aspects, surfactants (e.g., polysorbates)are employed as adjunct compounds, processing agents, or formulationadditives to enhance intranasal delivery of the peptide. Agents such asDMSO, polyethylene glycol, and ethanol can, if present in sufficientlyhigh concentrations in delivery environment (e.g., by pre-administrationor incorporation in a therapeutic formulation), enter the aqueous phaseof the mucosa and alter its solubilizing properties, thereby enhancingthe partitioning of the peptide from the vehicle into the mucosa. Themucosal therapeutic and prophylactic compositions of the presentinvention may be supplemented with any suitable penetration-promotingagent that facilitates absorption, diffusion, or penetration of thepeptide across mucosal barriers. The penetration promoter may be anypromoter that is pharmaceutically acceptable.

Charge Modifying and pH Control Agents and Methods

To improve the transport characteristics of biologically active agents(including the peptide within the present invention), for enhanceddelivery across hydrophobic mucosal membrane barriers, the inventionalso provides techniques and reagents for charge modification ofselected biologically active agents or delivery-enhancing agentsdescribed herein. In this regard, the relative permeabilities ofmacromolecules is generally be related to their partition coefficients.The degree of ionization of molecules, which is dependent on the pKa ofthe molecule and the pH at the mucosal membrane surface, also affectspermeability of the molecules. Permeation and partitioning ofbiologically active agents, including the peptide within the presentinvention, for mucosal delivery may be facilitated by charge alterationor charge spreading of the active agent or permeabilizing agent, whichis achieved, for example, by alteration of charged functional groups, bymodifying the pH of the delivery vehicle or solution in which the activeagent is delivered, or by coordinate administration of a charge- orpH-altering reagent with the active agent. Consistent with these generalteachings, mucosal delivery of charged macromolecular species, includingthe peptide within the present invention is substantially improved whenthe active agent is delivered to the mucosal surface in a substantiallyun-ionized, or neutral, electrical charge state.

Certain peptide and protein components of mucosal formulations for usewithin the invention will be charge modified to yield an increase in thepositive charge density of the peptide or protein. These modificationsextend also to cationization of peptide and protein conjugates, carriersand other delivery forms disclosed herein.

Degradative Enzyme Inhibitory Agents and Methods

Another excipient that may be included in a trans-mucosal preparation isa degradative enzyme inhibitor. Any inhibitor that inhibits the activityof an enzyme to protect the biologically active agent(s) may be usefullyemployed in the compositions and methods of the invention. Useful enzymeinhibitors for the protection of biologically active proteins andpeptides include, for example, soybean trypsin inhibitor, pancreatictrypsin inhibitor, chymotrypsin inhibitor and trypsin and chrymotrypsininhibitor isolated from potato (solanum tuberosum L.) tubers. Acombination or mixtures of inhibitors may be employed. The inhibitor(s)may be incorporated in or bound to a carrier, e.g., a hydrophilicpolymer, coated on the surface of the dosage form which is to contactthe nasal mucosa, or incorporated in the superficial phase of thesurface, in combination with the biologically active agent or in aseparately administered (e.g., pre-administered) formulation. Additionalenzyme inhibitors for use within the invention are selected from a widerange of non-protein inhibitors that vary in their degree of potency andtoxicity. As described in further detail below, immobilization of theseadjunct agents to matrices or other delivery vehicles, or development ofchemically modified analogues, may be readily implemented to reduce oreven eliminate toxic effects, when they are encountered. Among thisbroad group of candidate enzyme inhibitors for use within the inventionare organophosphorous inhibitors, such as diisopropylfluorophosphate(DFP) and phenylmethylsulfonyl fluoride (PMSF), which are potent,irreversible inhibitors of serine proteases (e.g., trypsin andchymotrypsin). Yet another type of enzyme inhibitory agent for usewithin the methods and compositions of the invention are amino acids andmodified amino acids that interfere with enzymatic degradation ofspecific therapeutic compounds.

The therapeutic agents of the invention can be used to treat disordersfor which modulation of GPCR-related signal transduction pathways isefficacious. For example, the peptides of the invention falling withinFormulas II, IV, and VI are used to treat disorders for which modulationof FPLR1 is efficacious. Examples of such peptides are depicted in SEQID NOs: 1-7, 14-16, 26-27, 32-38. These peptides are used to treat anydisease or condition that involves neutrophil (polymorphonuclearleukocyte, PMN)-dependent damage or neutrophil regulation. The peptidesof the invention are also used to treat disorders associated withTNFα-initiated cytokine activity in a subject.

The peptides of the invention falling within Formulas I, III, IV and Vare used to treat disorders for which modulation of MrgX2 isefficacious. Examples of such peptides are depicted in SEQ ID NOs: 5,8-13, 17-25, 28-35, 39-50. The peptides of the invention falling withinFormula III are used to treat disorders for which modulation of MrgX1 isefficacious. Examples of such peptides are depicted in SEQ ID NOs: 8,39-50. The peptides of the invention falling within Formulas I and IIare also used to treat disorders for which modulation of Mas isefficacious. Examples of such peptides are depicted in SEQ ID NOs:6-7,9-16, 18-25, 27-31.

The peptides of the invention falling within Formulas I, II, III, IV, Vand VI, such as for example, peptides as depicted in SEQ ID NOs: 1-50,are useful for the treatment of inflammatory diseases including but notlimited to gastritis, gout, gouty arthritis, arthritis, rheumatoidarthritis, inflammatory bowel disease, Crohn's disease, ulcerativecolitis, ulcers, chronic bronchitis, asthma, allergy, acute lung injury,pulmonary inflammation, airway hyper-responsiveness, vasculitis, septicshock and inflammatory skin disorders, including but not limited topsoriasis, atopic dermatitis, eczema.

The peptides of the invention falling within Formulas I, II, III, IV, Vand VI, such as for example, peptides as depicted in SEQ ID NOs:1-50,are also useful for the treatment of fibrotic conditions involvingtissue remodeling following inflammation or ischemia-reperfusion injury,including but not limited to endomyocardial fibrosis; mediastinalfibrosis; idiopathy pulmonary fibrosis; pulmonary fibrosis;retroperitoneal fibrosis; fibrosis of the spleen; fibrosis of thepancreas; hepatic fibrosis (cirrhosis); fibromatosis; granulomatous lungdisease; and glomerulonephritis

The peptides of the invention falling within Formulas I, II, IV, and VI,such as for example, peptides as depicted in SEQ ID NOs:1-7, 9-16,18-38, are also useful in the treatment of autoimmune disease, includingbut not limited to multiple sclerosis, psoriasis, rheumatoid arthritis,systemic lupus erythematosus, ulcerative colitis, Crohn's disease,transplant rejection, immune disorders associated with grafttransplantation rejection, benign lymphocytic angiitis, lupuserythematosus, Hashimoto's thyroiditis, primary myxedema, Graves'disease, pernicious anemia, autoimmune atrophic gastritis, Addison'sdisease, insulin dependent diabetes mellitis, Good pasture's syndrome,myasthenia gravis, pemphigus, sympathetic ophthalmia, autoimmuneuveitis, autoimmune hemolytic anemia, idiopathic thrombocytopenia,primary biliary cirrhosis, chronic action hepatitis, ulceratis colitis,Sjogren's syndrome, rheumatic disease, polymyositis, scleroderma, mixedconnective tissue disease, inflammatory rheumatism, degenerativerheumatism, extra-articular rheumatism, collagen diseases, chronicpolyarthritis, psoriasis arthropathica, ankylosing spondylitis, juvenilerheumatoid arthritis, periarthritis humeroscapularis, panarteriitisnodosa, progressive systemic scleroderma, arthritis uratica,dermatomyositis, muscular rheumatism, myositis, myogelosis andchondrocalcinosis.

The peptides of the invention falling within Formulas I, II, IV, and VI,such as for example, peptides as depicted in SEQ ID NOs: 1-7, 9-16,18-38, are also useful in treating cardiovascular diseases and theircomplications, peripheral vascular diseases and coronary arterydiseases, including but not limited to myocardial infarction; congestiveheart failure (CHF); myocardial failure; myocardial hypertrophy;ischemic cardiomyopathy; systolic heart failure; diastolic heartfailure; stroke; thrombotic stroke; concentric LV hypertrophy,myocarditis; cardiomyopathy; hypertrophic cardiomyopathy; myocarditis;decompensated heart failure; ischemic myocardial disease; congenitalheart disease; angina pectoris; prevention of heart remodeling orventricular remodeling after myocardial infarction; ischemia-reperfusioninjury in ischemic and post-ischemic events (e.g. myocardial infarct);cerebrovascular accident; mitral valve regurgitation; hypertension;hypotension; restenosis; fibrosis; thrombosis; or platelet aggregation.

The peptides of the invention falling within Formulas I, II, IV, and VI,such as for example, peptides as depicted in SEQ ID NOs: 1-7, 9-16,18-38, are useful in treating ischemia-reperfusion injury associatedwith ischemic and post-ischemic events in organs and tissues, includingbut not limited to thrombotic stroke; myocardial infarction; anginapectoris; embolic vascular occlusions; peripheral vascularinsufficiency; splanchnic artery occlusion; arterial occlusion bythrombi or embolisms, arterial occlusion by non-occlusive processes suchas following low mesenteric flow or sepsis; mesenteric arterialocclusion; mesenteric vein occlusion; ischemia-reperfusion injury to themesenteric microcirculation; ischemic acute renal failure;ischemia-reperfusion injury to the cerebral tissue; intestinalintussusception; hemodynamic shock; tissue dysfunction; organ failure;restenosis; atherosclerosis; thrombosis; platelet aggregation.

The peptides of the invention falling within Formulas I, II, IV, and VI,such as for example, peptides as depicted in SEQ ID NOs: 1-7, 9-16,18-38, are useful in treating ischemia-reperfusion injury followingconditions including but not limited to procedures such as cardiacsurgery; organ surgery; organ transplantation; angiography;cardiopulmonary and cerebral resuscitation.

The peptides of the invention falling within Formulas I, II, IV, and VI,such as for example, peptides as depicted in SEQ ID NOs: 1-7, 9-16,18-38, are useful in the inhibition of alopecia, such aschemotherapy-induced alopecia; and treatment of bone disease, such asosteoporosis.

In another aspect, the peptides of the invention falling within FormulasI, II, III, IV, and V, such as for example, peptides as depicted in SEQID NOs:5-25, 27-35, 39-50, are used for prevention and treatment ofhypertension and its complications including but not limited tohypertensive heart disease; antihypertension (blood pressure reduction);systemic and pulmonary high blood pressure; cerebrovascular disease andstroke; heart failure and stroke; left ventricular hypertrophy (LVH);congestive heart failure (CHF); hypertension, high blood pressure;vasodilation; renal hypertension; diuresis; nephritis; natriuresis;scleroderma renal crisis; angina pectoris (stable and unstable);myocardial infarction; heart attack; coronary artery disease; coronaryheart disease; cardiac arrhythmias; atrial fibrillation; portalhypertension; raised intraocular pressure; vascular restenosis; chronichypertension; valvular disease; myocardial ischemia; acute pulmonaryedema; acute coronary syndrome; hypertensive retinopathy; hypertensivepregnancy sickness; preeclampsia; Raynaud's phenomenon; erectiledysfunction and glaucoma. These peptides are also used as a vasodilatorand in antithrombotic therapy.

The peptides of the invention falling within Formulas I, II, IV, and VI,such as for example, peptides as depicted in SEQ ID NOs: 1-7, 9-16,18-38, are also useful in treating inflammatory conditions associatedwith an infection, e.g., a bacterial infection or a viral infection,including but not limited to a viral infection caused by humanimmunodeficiency virus I (HIV-1) or HIV-2, acquired immune deficiency(AIDS), West Nile encephalitis virus, coronavirus, rhinovirus, influenzavirus, dengue virus, hemorrhagic fever; an otological infection; severeacute respiratory syndrome (SARS), sepsis and sinusitis.

The peptides of the invention falling within Formulas I, II, IV, and VI,such as for example, peptides as depicted in SEQ ID NOs: 1-7, 9-16,18-38, are also useful in the prevention or treatment of cancer, orinflammation associated with cancer such as solid cancer, including butnot limited to colon cancer, lung cancer, breast cancer, prostatecancer, brain cancer, pancreatic cancer, ovarian cancer or kidneycancer. The cancer can alternatively be a melanoma, glioma, a sarcoma, aleukemia, or lymphoma. These peptides are also useful in the preventionor treatment of invasive and metastatic cancer.

The peptides of the invention falling within Formulas I and II, such asfor example, peptides as depicted in SEQ ID NOs:6-7, 9-16, 18-25, 27-31,are used in the prevention and treatment of diseases that involvereduction of oxygen reactive species with consequent endothelialdysfunction, including but not limited to cardiovascular diseases, highblood pressure, atherosclerosis, thrombosis, myocardial infarct, heartfailure, renal diseases, plurimetabolic syndrome, erectile dysfunction;vasculitis; and diseases of the central nervous system (CNS).

In another aspect, the peptides of the invention falling within FormulasI and II, such as for example, peptides as depicted in SEQ ID NOs:6-7,9-16, 18-25, 27-31, are used in the prevention and/or treatment oforganic alterations produced by aging and as ergogenic aids.

The peptides of the invention falling within Formulas I and II, such asfor example, peptides as depicted in SEQ ID NOs:6-7, 9-16, 18-25, 27-31,are used in the prevention and treatment of diseases that involvealterations in the muscular differentiation, maturation and regenerationin muscular atrophies, including but not limited to as cachexia;prolonged restriction to bed due to numerous factors; chronic use ofcorticoids; and varied neurological syndromes, traumatisms anddegenerative diseases that lead to muscular atrophy. The peptides of theinvention are used for the prevention or treatment of organicalterations produced by aging and as ergogenic aids.

The peptides of the invention falling within Formulas I, II, IV and VI,such as for example, peptides as depicted in SEQ ID NOs: 1-7, 9-16,18-38, are used for the prevention and treatment in skin injuries,including but not limited to dermal repair, wound healing; burns,erythemas, lesions, and skin tumors.

The peptides of the invention falling within Formulas I, II, IV and VI,such as for example, peptides as depicted in SEQ ID NOs: 1-7, 9-16,18-38, are used for the prevention or treatment of immune relatedconditions including but not limited to graft versus host disease;transplant rejection, bone marrow transplantation.

The peptides of the invention falling within Formulas II, IV and VI,such as for example, peptides as depicted in SEQ ID NOs:1-7, 14-16,26-27, 32-38, are used for mobilization, activation or inducingchemoattraction of blood cells to a site of injury. The blood cells caninclude platelets, phagocytes, monocytes, macrophages, eosinophils,neutrophils, and/or lymphocytes.

The peptides of the invention falling within Formulas I and II, such asfor example, peptides as depicted in SEQ ID NOs:6-7, 9-16, 18-25, 27-31,are used for prevention or treatment of genetic polymorphism consequentdiseases such as the DD type of the angiotensin converting enzyme; typeI and type II diabetes mellitus and complications; diabetic mellitusprophylaxis; diabetic maculopathy; and diabetic nephropathy.

The peptides of the invention falling within Formulas I and II, such asfor example, peptides as depicted in SEQ ID NOs:6-7, 9-16, 18-25, 27-31,are used in the prevention or treatment of a urogenital disorder or agenitor-urological disorders including but not limited to renal disease;a bladder disorder; disorders of the reproductive system; gynecologicdisorders; urinary tract disorder; incontinence; disorders of the male(spermatogenesis, spermatic motility), and female reproductive system;sexual dysfunction; erectile dysfunction; embryogenesis; and pregnancyrelated disorders. These are also used in pregnancy monitoring.

The peptides of the invention falling within Formulas I, II, IV and VI,such as for example, peptides as depicted in SEQ ID NOs: 1-7, 9-16,18-38, are used in the prevention or treatment of a cytopenia, includingbut not limited to a multilineage cytopenia, a thrombocytopenia, anemia,anemia due to renal failure; lymphopenia, leucopenia, neutropenia,radio/chemotherapy-related neutropenia; and platelet disorders.

The invention also provides peptides falling within Formulas I, II, IVand VI, such as for example, peptides as depicted in SEQ ID NOs: 1-7,9-16, 18-38, that are used in the prevention or treatment of respiratorydiseases, including but not limited to asthma, bronchial disease, lungdiseases, chronic obstructive pulmonary disease (COPD), AcuteRespiratory Distress Syndrome (ARDS), severe acute respiratory syndrome(SARS)

The invention also provides peptides falling within Formulas I, II, IV,and VI, such as for example, peptides as depicted in SEQ ID NOs: 1-7,9-16, 18-38, that are used in the prevention or treatment of metabolicdisorders including but not limited to diabetes, diabetis mellitus,lipodystrophy, hyperthyroidism, glaucoma, hyperlipidaemia, non-insulindependent diabetes, appetite control and obesity.

The peptides of the invention falling within Formulas I, II, III, IV,and V, such as for example, peptides as depicted in SEQ ID NOs:5-25,27-35, 39-50, are also used in the prevention and treatment of kidneydiseases including but not limited to diabetic nephropathy;glomerulosclerosis; nephropathies; renal impairment; scleroderma renalcrisis and chronic renal failure. These peptides can also be used asantidiuretics.

The peptides of the invention falling within Formulas I and II, such asfor example, peptides as depicted in SEQ ID NOs:6-7, 9-16, 18-25, 27-31,are also used in the prevention and treatment of blood diseasesincluding but not limited to angioplasty (endoluminal prosthesis andpost angioplasty restenosis); haematopoiesis; erythrocytosis; disordersof the blood crasis, such as post radiotherapy.

The peptides of the invention falling within Formulas I, II, IV, and VI,such as for example, peptides as depicted in SEQ ID NOs: 1-7, 9-16,18-38, are also used in the prevention and treatment of angiogenesisrelated conditions including but not limited to retinal angiogenesis ina number of human ocular diseases such as diabetes mellitus, retinopathyof prematury, and age-related macular degeneration, or cancer associatedangiogenesis in primary or metastatic cancer, including but not limitedto cancer of the prostate, brain, breast, colorectal, lung, ovarian,pancreatic, renal, cervical, melanoma, soft tissue sarcomas, lymphomas,head-and-neck, and glioblastomas.

The peptides of the invention falling within Formulas I, II, III, IV,and V, such as for example, peptides as depicted in SEQ ID NOs:5-25,27-35, 39-50, are also used to treat a central nervous system (CNS)disorder, including but not limited to central and peripheraldegenerative neuropathies; neuroprotection; impaired cognition; anxietydisorders, pain control, food intake, a behavioral disorder, a learningdisorder, a sleep disorder, a memory disorder, a pathologic response toanesthesia, addiction, depression, migraine, a menstruation disorder,muscle spasm, opiate dependence, dementia, Alzheimer's disease,Parkinson's disease, cortical function, locomotor activity and aperipheral nervous system disorder.

The peptides of the invention falling within Formulas I, III, IV, and V,such as for example, peptides as depicted in SEQ ID NOs:5, 8-13, 17-25,28-35, 39-50, are also used to treat and control pain including but notlimited to complex regional pain, muscoskeletal pain, neuropathic pain,post-herpetic pain, pain associated with cancer, or post-operative pain.

Compounds of Formula I, II, III, IV, V, or VI can be used to treatdisorders, diseases and/or conditions as described herein, byadministering to a subject in need thereof a therapeutically effectiveamount of a peptide falling within Formula I, II, III, IV, V, or VI.

Also provided by the invention is a method of treating disorders forwhich modulation of GPCR-related signal transduction pathways isefficacious. For example, provided by the invention is a method oftreating disorders for which modulation of FPLR1 is efficacious in asubject by administering to a subject in need thereof a therapeuticallyeffective amount of a compound falling within Formulas II, IV, and VI,such as for example, peptides as depicted in SEQ ID NOs: 1-7, 9-16,18-38. Such diseases disorders and/or condition can involves neutrophil(polymorphonuclear leukocyte, PMN)-dependent damage or neutrophilregulation, and/or disorders associated with TNFα-initiated cytokineactivity in a subject.

Also provided by the invention is a method of treating disorders forwhich modulation of MrgX2 is efficacious in a subject by administeringto a subject in need thereof a therapeutically effective amount of acompound falling within Formulas I, III, IV and V such as for example,peptides as depicted in SEQ ID NOs: 5, 8-13, 17-25, 28-35, 39-50.

Also provided by the invention is a method of treating disorders forwhich modulation of MrgX1 is efficacious in a subject by administeringto a subject in need thereof a therapeutically effective amount of acompound falling within Formula III, such as for example, peptides asdepicted in SEQ ID NOs: 8, 39-50.

Also provided by the invention is a method of treating treat disordersfor which modulation of Mas is efficacious in a subject by administeringto a subject in need thereof a therapeutically effective amount of acompound falling within Formulas I and II, such as for example, peptidesas depicted in SEQ ID NOs.6-7, 9-16, 18-25, 27-31.

Also provided by the invention is a method of treating an inflammatorydisorder in a subject by administering to a subject in need thereof atherapeutically effective amount of a compound falling within Formula I,II, III, IV, V and VI, such as for example, peptides as depicted in SEQID NOs: 1-50. The inflammatory disorder can be gastritis, gout, goutyarthritis, arthritis, rheumatoid arthritis, inflammatory bowel disease,Crohn's disease, ulcerative colitis, ulcers, chronic bronchitis, asthma,allergy, acute lung injury, pulmonary inflammation, airwayhyper-responsiveness, vasculitis, septic shock and inflammatory skindisorders, including but not limited to psoriasis, atopic dermatitis,eczema.

Also provided by the invention is a method of treating fibroticconditions involving tissue remodeling following inflammation orischemia-reperfusion injury in a subject by administering to a subjectin need thereof a therapeutically effective amount of a compound fallingwithin Formula I, II, III, IV, V and VI, such as for example, peptidesas depicted in SEQ ID NOs:1-50. These fibrotic conditions can beendomyocardial fibrosis; mediastinal fibrosis; idiopathy pulmonaryfibrosis; pulmonary fibrosis; retroperitoneal fibrosis; fibrosis of thespleen; fibrosis of the pancreas; hepatic fibrosis (cirrhosis);fibromatosis; granulomatous lung disease; and glomerulonephritis.

Also provided by the invention is a method of treating an autoimmunedisease or disorder in a subject by administering to a subject in needthereof a therapeutically effective amount of a compound falling withinFormula I, II, IV, and VI, such as for example, peptides as depicted inSEQ ID NOs: 1-7, 9-16, 18-38. The autoimmune disease can be multiplesclerosis, psoriasis, rheumatoid arthritis, systemic lupuserythematosus, ulcerative colitis, Crohn's disease, transplantrejection, immune disorders associated with graft transplantationrejection, benign lymphocytic angiitis, lupus erythematosus, Hashimoto'sthyroiditis, primary myxedema, Graves' disease, pernicious anemia,autoimmune atrophic gastritis, Addison's disease, insulin dependentdiabetes mellitis, Good pasture's syndrome, myasthenia gravis,pemphigus, sympathetic ophthalmia, autoimmune uveitis, autoimmunehemolytic anemia, idiopathic thrombocytopenia, primary biliarycirrhosis, chronic action hepatitis, ulceratis colitis, Sjogren'ssyndrome, rheumatic disease, polymyositis, scleroderma, mixed connectivetissue disease, inflammatory rheumatism, degenerative rheumatism,extra-articular rheumatism, collagen diseases, chronic polyarthritis,psoriasis arthropathica, ankylosing spondylitis, juvenile rheumatoidarthritis, periarthritis humeroscapularis, panarteriitis nodosa,progressive systemic scleroderma, arthritis uratica, dermatomyositis,muscular rheumatism, myositis, myogelosis and chondrocalcinosis.

Also provided by the invention is a method of treating cardiovasculardiseases and their complications in a subject by administering to asubject in need thereof a therapeutically effective amount of a compoundfalling within Formula I, II, IV, and VI, such as for example, peptidesas depicted in SEQ ID NOs: 1-7, 9-16, 18-38. The cardiovascular diseasescan be peripheral vascular diseases and coronary artery diseases,including but not limited to myocardial infarction; coronary heartdisease; congestive heart failure (CHF); myocardial failure; myocardialhypertrophy; ischemic cardiomyopathy; systolic heart failure; diastolicheart failure; stroke; thrombotic stroke; concentric LV hypertrophy,myocarditis; cardiomyopathy; hypertrophic cardiomyopathy; myocarditis;decompensated heart failure; ischemic myocardial disease; congenitalheart disease; angina pectoris; prevention of heart remodeling orventricular remodeling after myocardial infarction; ischemia—reperfusioninjury in ischemic and post-ischemic events (e.g. myocardial infarct);cerebrovascular accident; mitral valve regurgitation; hypertension;hypotension; restenosis; fibrosis; thrombosis; or platelet aggregation.

Also provided by the invention is a method of treating anischemia-reperfusion injury in a subject by administering to a subjectin need thereof a therapeutically effective amount of a compound fallingwithin Formula I, II, IV, and VI, such as for example, peptides asdepicted in SEQ ID NOs: 1-7, 9-16, 18-38. The ischemia-reperfusioninjury can be associated with ischemic and post-ischemic events inorgans and tissues, including but not limited to thrombotic stroke;myocardial infarction; angina pectoris; embolic vascular occlusions;peripheral vascular insufficiency; splanchnic artery occlusion; arterialocclusion by thrombi or embolisms, arterial occlusion by non-occlusiveprocesses such as following low mesenteric flow or sepsis; mesentericarterial occlusion; mesenteric vein occlusion; ischemia-reperfusioninjury to the mesenteric microcirculation; ischemic acute renal failure;ischemia-reperfusion injury to the cerebral tissue; intestinalintussusception; hemodynamic shock; tissue dysfunction; organ failure;restenosis; atherosclerosis; thrombosis; platelet aggregation. Theischemia-reperfusion injury can be alternatively following conditionsincluding but not limited to procedures such as cardiac surgery; organsurgery; organ transplantation; angiography; cardiopulmonary andcerebral resuscitation.

Also provided by the invention is a method of treating other variousdisorders, diseases and/or conditions in a subject by administering to asubject in need thereof a therapeutically effective amount of a compoundfalling within Formula I, II, IV, and VI, such as for example, peptidesas depicted in SEQ ID NOs: 1-7, 9-16, 18-38. Such disorders, diseasesand/or conditions can be inhibition of alopecia, such as chemotherapy-induced alopecia; or treatment of bone disease, such as osteoporosis.

Also provided by the invention is a method of preventing and treating anhypertension and its complications in a subject by administering to asubject in need thereof a therapeutically effective amount of a compoundfalling within Formula I, II, III, IV, and V, such as for example,peptides as depicted in SEQ ID NOs:5-25, 27-35, 39-50. The hypertensionand its complications can be hypertensive heart disease;antihypertension (blood pressure reduction); systemic and pulmonary highblood pressure; cerebrovascular disease and stroke; heart failure andstroke; left ventricular hypertrophy (LVH); congestive heart failure(CHF); hypertension, high blood pressure; vasodilation; renalhypertension; diuresis; nephritis; natriuresis; scleroderma renalcrisis; angina pectoris (stable and unstable); myocardial infarction;heart attack; coronary artery disease; coronary heart disease; cardiacarrhythmias; atrial fibrillation; portal hypertension; raisedintraocular pressure; vascular restenosis; chronic hypertension;valvular disease; myocardial ischemia; acute pulmonary edema; acutecoronary syndrome; hypertensive retinopathy; hypertensive pregnancysickness; preeclampsia; Raynaud's phenomenon; erectile dysfunction andglaucoma. These peptides are also used as a vasodilator and inantithrombotic therapy.

Also provided by the invention is a method of treating an inflammatorydisorder and/or conditions associated with an infection in a subject byadministering to a subject in need thereof a therapeutically effectiveamount of a compound falling within Formulas I, II, IV, and VI, such asfor example, peptides as depicted in SEQ ID NOs: 1-7, 9-16, 18-38. Theinflammatory conditions associated with an infection, can be a bacterialinfection or a viral infection, including but not limited to a viralinfection caused by human immunodeficiency virus I (HIV-1) or HIV-2,acquired immune deficiency (AIDS), West Nile encephalitis virus,coronavirus, rhinovirus, influenza virus, dengue virus, hemorrhagicfever; an otological infection; severe acute respiratory syndrome(SARS), sepsis and sinusitis.

Also provided by the invention is a method of treating cancer, orinflammation associated with cancer in a subject by administering to asubject in need thereof a therapeutically effective amount of a compoundfalling within Formulas I, II, IV, and VI, such as for example, peptidesas depicted in SEQ ID NOs: 1-7, 9-16, 18-38. The cancer, or inflammationassociated with cancer can be solid cancer, including but not limited tocolon cancer, lung cancer, breast cancer, prostate cancer, brain cancer,pancreatic cancer, ovarian cancer or kidney cancer. The cancer canalternatively be a melanoma, glioma, a sarcoma, a leukemia, or lymphoma.These peptides are also useful in the prevention or treatment ofinvasive and metastatic cancer.

Also provided by the invention is a method of treating of diseases thatinvolve reduction of oxygen reactive species with consequent endothelialdysfunction in a subject by administering to a subject in need thereof atherapeutically effective amount of a compound falling within Formulas Iand II, such as for example, peptides as depicted in SEQ ID NOs:6-7,9-16, 18-25, 27-31. The diseases that involve reduction of oxygenreactive species with consequent endothelial dysfunction, can becardiovascular diseases, high blood pressure, atherosclerosis,thrombosis, myocardial infarct, heart failure, renal diseases,plurimetabolic syndrome, erectile dysfunction; vasculitis; and diseasesof the central nervous system (CNS).

Also provided by the invention is a method of prevention and/ortreatment of organic alterations produced by aging in a subject byadministering to a subject in need thereof a therapeutically effectiveamount of a compound falling within Formulas I and II, such as forexample, peptides as depicted in SEQ ID NOs:6-7, 9-16, 18-25, 27-31.

Also provided by the invention is a method of prevention and treatmentof diseases that involve alterations in the muscular differentiation,maturation and regeneration in muscular atrophies in a subject byadministering to a subject in need thereof a therapeutically effectiveamount of a compound falling within Formulas I and II, such as forexample, peptides as depicted in SEQ ID NOs:6-7, 9-16, 18-25, 27-31. Thediseases that involve alterations in the muscular differentiation,maturation and regeneration in muscular atrophies, including but notlimited to as cachexia; prolonged restriction to bed due to numerousfactors; chronic use of corticoids; and varied neurological syndromes,traumatisms and degenerative diseases that lead to muscular atrophy. Thepeptides of the invention are used for the prevention or treatment oforganic alterations produced by aging and as ergogenic aids.

Also provided by the invention is a method of treating skin injurydiseases, disorders and/or conditions in a subject by administering to asubject in need thereof a therapeutically effective amount of a compoundfalling within Formulas I, II, IV and VI, such as for example, peptidesas depicted in SEQ ID NOs: 1-7, 9-16, 18-38. The skin injury diseases,disorders and/or conditions can be dermal repair, wound healing; burns,erythemas, lesions, and skin tumors.

Also provided by the invention is a method of prevention or treatment ofimmune related conditions in a subject by administering to a subject inneed thereof a therapeutically effective amount of a compound fallingwithin Formulas I, II, IV and VI, such as for example, peptides asdepicted in SEQ ID NOs: 1-7, 9-16, 18-38. The immune related conditionsincluding but not limited to graft versus host disease; transplantrejection, bone marrow transplantation.

Also provided by the invention is a method of mobilization, activationor inducing chemoattraction of blood cells to a site of injury in asubject by administering to a subject in need thereof a therapeuticallyeffective amount of a compound falling within Formulas II, IV and VI,such as for example, peptides as depicted in SEQ ID NOs:1-7, 14-16,26-27, 32-38. The blood cells can include platelets, phagocytes,monocytes, macrophages, eosinophils, neutrophils, and/or lymphocytes.

Also provided by the invention is a method of treating geneticpolymorphism consequent diseases in a subject by administering to asubject in need thereof a therapeutically effective amount of a compoundfalling within Formulas I and II, such as for example, peptides asdepicted in SEQ ID NOs:6-7, 9-16, 18-25, 27-31. The genetic polymorphismconsequent diseases can be DD type of the angiotensin converting enzyme;type I and type II diabetes mellitus and complications; diabeticmellitus prophylaxis; diabetic maculopathy; and diabetic nephropathy.

Also provided by the invention is a method of prevention or treatment ofa urogenital disorder or a genitor-urological disorders in a subject byadministering to a subject in need thereof a therapeutically effectiveamount of a compound falling within Formulas I and II, such as forexample, peptides as depicted in SEQ ID NOs:6-7, 9-16, 18-25, 27-31. Thea urogenital disorder or a genitor-urological disorders can be renaldisease; a bladder disorder; disorders of the reproductive system;gynecologic disorders; urinary tract disorder; incontinence; disordersof the male (spermatogenesis, spermatic motility), and femalereproductive system; sexual dysfunction; erectile dysfunction;embryogenesis; and pregnancy related disorders. These are also used inpregnancy monitoring.

Also provided by the invention is a method of treating cytopenia in asubject by administering to a subject in need thereof a therapeuticallyeffective amount of a compound falling within Formulas I, II, IV and VI,such as for example, peptides as depicted in SEQ ID NOs: 1-7, 9-16,18-38. The cytopenia can be multilineage cytopenia, a thrombocytopenia,anemia, anemia due to renal failure; lymphopenia, leucopenia,neutropenia, radio/chemotherapy-related neutropenia; and plateletdisorders.

Also provided by the invention is a method of prevention or treatment ofrespiratory diseases in a subject by administering to a subject in needthereof a therapeutically effective amount of a compound falling withinFormulas I, II, IV and VI, such as for example, peptides as depicted inSEQ ID NOs: 1-7, 9-16, 18-38. The respiratory diseases can be asthma,bronchial disease, lung diseases, chronic obstructive pulmonary disease(COPD), Acute Respiratory Distress Syndrome (ARDS), severe acuterespiratory syndrome (SARS).

Also provided by the invention is a method of prevention or treatment ofmetabolic disorders in a subject by administering to a subject in needthereof a therapeutically effective amount of a compound falling withinFormulas I, II, IV, and VI, such as for example, peptides as depicted inSEQ ID NOs: 1-7, 9-16, 18-38. The metabolic disorders can be diabetes,diabetis mellitus, lipodystrophy, hyperthyroidism, glaucoma,hyperlipidaemia, non-insulin dependent diabetes, appetite control andobesity.

Also provided by the invention is a method of prevention and treatmentof kidney diseases in a subject by administering to a subject in needthereof a therapeutically effective amount of a compound falling withinI, II, III, IV, and V, such as for example, peptides as depicted in SEQID NOs:5-25, 27-35, 39-50. The kidney diseases can be diabeticnephropathy; glomerulosclerosis; nephropathies; renal impairment;scleroderma renal crisis and chronic renal failure.

Also provided by the invention is a method of prevention and treatmentof blood diseases in a subject by administering to a subject in needthereof a therapeutically effective amount of a compound falling withinFormula I and II, such as for example, peptides as depicted in SEQ IDNOs:6-7, 9-16, 18-25, 27-31. The blood diseases can be angioplasty(endoluminal prosthesis and post angioplasty restenosis);haematopoiesis; erythrocytosis; disorders of the blood crasis, such aspost radiotherapy.

Also provided by the invention is a method of prevention and treatmentof angiogenesis related conditions in a subject by administering to asubject in need thereof a therapeutically effective amount of a compoundfalling within Formulas I, II, IV, and VI, such as for example, peptidesas depicted in SEQ ID NOs: 1-7, 9-16, 18-38. The angiogenesis relatedconditions including but not limited to retinal angiogenesis in a numberof human ocular diseases such as diabetes mellitus, retinopathy ofprematury, and age-related macular degeneration, or cancer associatedangiogenesis in primary or metastatic cancer, including but not limitedto cancer of the prostate, brain, breast, colorectal, lung, ovarian,pancreatic, renal, cervical, melanoma, soft tissue sarcomas, lymphomas,head-and-neck, and glioblastomas.

Also provided by the invention is a method of treating central nervoussystem (CNS) disorder, in a subject by administering to a subject inneed thereof a therapeutically effective amount of a compound fallingwithin Formulas I, II, III, IV, and V, such as for example, peptides asdepicted in SEQ ID NOs:5-25, 27-35, 39-50. The central nervous system(CNS) disorder, including but not limited to central and peripheraldegenerative neuropathies; neuroprotection; impaired cognition; anxietydisorders, pain control, food intake, a behavioral disorder, a learningdisorder, a sleep disorder, a memory disorder, a pathologic response toanesthesia, addiction, depression, migraine, a menstruation disorder,muscle spasm, opiate dependence, dementia, Alzheimer's disease,Parkinson's disease, cortical function, locomotor activity and aperipheral nervous system disorder.

Also provided by the invention is a method of treating and controlingpain in a subject by administering to a subject in need thereof atherapeutically effective amount of a compound falling within FormulasI, III, IV, and V, such as for example, peptides as depicted in SEQ IDNOs:5, 8-13, 17-25, 28-35, 39-50. The pain includes but not limited tocomplex regional pain, muscoskeletal pain, neuropathic pain,post-herpetic pain, pain associated with cancer, or post-operative pain.

Optionally, the cDNA that encodes the peptide sequences of the inventionare used in gene therapy to treat the respective diseases, disordersand/or conditions, as detailed hereinabove.

The invention will be further illustrated in the following examples.

Method for analysis of peptides' ability to influence Calcium Flux(Revelant to Examples 1-4, below)

The assay was carried out in CHO cells were transiently transfected withthe GPCR of choice, by utilizing the promiscuous Gα16 to divertsignaling to the Gq pathway, thus enabling readout of GPCR activation bytesting for Calcium flux as described by Liu et al 2003 (Biomol Screen8, 39-49).

Peptides (listed in FIG. 1) were synthesized by the solid phase peptidesynthesis (SPPS) method, cleaved from the resin, and purified by RP-HPLCunless stated otherwise. The peptide's identity was verified by massspectrometry. Final purity of peptide was >90% as measured by RP-HPLC.Peptides were diluted in PBS containing 0.1% BSA. All plates were storedat −80 C until use.

All the peptides thus obtained were tested for their ability to changecalcium flux in CHO-K1 cells (ATCC-CCL-61) transiently co-transfectedwith an expression vector containing the GPCR of choice and expressionvector containing the Gα16 encoding cDNA. Expression constructscontaining cDNA clones for MrgX1, MrgX2, Mas or FPRL1 were commerciallyobtained in one of the following expression vectors: pcDNA3.1, pCMV6, orMO2.

Transient transfections were performed using CHO-K1 cells as host cells.Cells (12 million) were plated into T75 flasks on the day precedingtransfection. Cells were transfected with the appropriate GPCRexpression vector and with a vector expressing G_(α16) using alipid-based reagent, MTI, according to the manufacturer'srecommendation. Cells were transfected for 5 hours, then re-plated into96-well dishes (60,000 cells per well) and grown overnight.

On the day of the experiment, cells were loaded with Fluo-4-NW(Invitrogen) according to the manufacturer's recommendation. Plates wereloaded into a FlexStation™ (Molecular Devices) plate reader andfluorescence was monitored. Seventeen seconds following initiation ofreading, cells were stimulated with the indicated agonist/compound atfinal concentration of 1 μM. Each 96 well plate contained cellstransfected with one GPCR expression vector and each of the examinedpeptides was tested in triplicate.

We defined a hit as a peptide which elicited a clear and distinctincrease in intracellular calcium that is clearly visible andstatistically significant upon examination of the calcium trace for atleast two repeats. The criterion for statistical significance was ap-value lower than 0.001 in a t-test comparing the levels of calciumbefore and after peptide addition.

Based on dose response experiments, EC50 best fit values were calculatedby non-linear regression of sigmoidal dose-response curves, using Prismversion 4 (GraphPad Software Inc., San Diego, Calif.). The formulae forthe sigmoidal dose-response curves was defined asY=Bottom+(Top−Bottom)/(1+10^(Log EC50−X)*HillSlope))

EXAMPLE 1 Induction of Calcium Flux in MrgX1-Transfected CHO Cells

The ability of Peptides to change calcium flux was examined as describedabove in CHO-K1 cells co-transfected with MrgX1 and Gα16. The followingresults were obtained: Peptide 60_S (SEQ ID NO:8)

As shown in FIG. 2, P60_S (SEQ ID NO:8) present in all three wellsincreased calcium flux of MrgX1-transfected CHO cells during the timeperiod between 20 seconds and 90 seconds relative to the negativecontrol. In addition, P60_S induced a dose dependent activation of MrgX1(FIG. 3) when examined at 1, 3, 10, 30, 100, 300, 1000, and 3000 nM. Theresponse to P60_S at the highest concentration was similar to that ofthe positive control, BAM22 (FIG. 3). From these results, the EC50 wascalculated to be at least 300 nM for Peptide 60_S (SEQ ID NO:8), whilethe EC50 for BAM22 is at least 50 nM.

EXAMPLE 2 Induction of Calcium Flux in MrgX2-Transfected CHO Cells

The ability of Peptides to change calcium flux was examined in CHO-K1cells co-transfected with MrgX2 and Gα16. Several peptides were found toinduce calcium flux in this experimental system as follows:

Peptide 60_S (SEQ ID NO:8)

As shown in FIG. 4, the sample of P60_S (SEQ ID NO:8) present in allthree wells increased calcium flux during the time period between about20 seconds and 90 seconds relative to the negative control.

Peptide 94 (SEQ ID NO:5)

As shown in FIG. 5 the sample of P94 (SEQ ID NO:5) present in all threewells increased calcium flux during the time period between about 20seconds and 90 seconds relative to the negative control.

Peptide 61_S (SEQ ID NO:10)

As shown in FIG. 6, the samples of Peptide 61_S (SEQ ID NO:10) presentin all three wells increased calcium flux during the time period betweenabout 20 seconds and 90 seconds relative to the negative control.

Peptide 63 (SEQ ID NO:17)

As shown in FIG. 7, the sample of Peptide 63 (SEQ ID NO:17) present inboth wells increased calcium flux during the time period between about20 seconds and 90 seconds relative to the negative control.

EXAMPLE 3 Induction of Calcium Flux in Mas-Transfected CHO Cells

The ability of Peptides to change calcium flux was examined in CHO-K1cells co-transfected with Mas receptor and Gα16. The following peptideswere found to induce calcium flux in this experimental system:

A. Activation of Mas by P33 Family

a. P33

As detailed in FIG. 1, Peptide 33 (SEQ ID NO:6) contains a Cysteine inposition 3. In order to avoid dimerization or other interaction of thepeptide via the Cysteine, this amino acid was substituted for Val tocreate P33_V (SEQ ID NO:7) which allowed the synthesis of a homogenouslymonomeric peptide. As shown in FIG. 8, the sample of P33_V (SEQ ID NO:7)present in well 2 increased calcium flux in the transfected cells duringthe time period between 20 seconds and 90 seconds relative to thenegative control. The samples of P33_V (SEQ ID NO:7) present in wells 1and 3 increased calcium flux during the time period between 60 secondsand 90 seconds relative to the negative control.

FIG. 39 shows the dose response of Mas to Peptide 33_V (SEQ ID NO. 7).The doses examined in this experiment were 10, 30, 100, 300, 1000 and3000 nM. The known endogenous Mas ligand, Ang (1-7) (data not-shown),did not induce Ca response in this assay, as reported in the literature(Santos et al., (2003), PNAS: 100, 8258-8263). The highest response toPeptide 33_V was not reached. The results indicate that the EC50 forPeptide 33_V is at least 1000 nM.

b. P33 Peptide Derivatives

In order to further characterize the activity of P33, the original, Cysbearing P33, was synthesized both as a monomer and a dimer (P33_mono andP33_dimer also named P33_D, respectively; (SEQ ID NO. 6)). The monomericand dimeric forms of P33 were synthesized by the solid phase peptidesynthesis (SPPS) with the “FMOC” strategy. The crude peptides were thenobtained by cleavage form the resin by TFA solution. Purification of thecrude peptides was done on HPLC to the degree of 95%. Dimerization ofthe Cysteine was performed by air oxidation and the dimer was againpurified to 95%. To avoid dimerization or other interaction of a peptidethat contains Cysteine (P33_mono), the sulfhydryl moiety of the Cysteinewas protected with Acm group which remains stable under the acidiccleavage of the peptide from resin. For P33_V, the TFA salt was furtherreplaced to acetate salt with the aid of ammoniumacetate and acetic acidfollowed by HPLC purification. The identity of the peptides wasdetermined by Mass Spectroscopy.

In addition, shorter derivates of P33 (P33_(—)5, P33_(—)8, P33_(—)9,P33_(—)10; SEQ ID NOs:27, 14, 15, 16, respectively, FIG. 1) weresynthesized and studied for their ability to induce calcium flux in theMas-transfected CHO-K1 cells. The shorter derivates of Peptide 33 weresynthesized by the solid phase peptide synthesis (SPPS) with the “FMOC”strategy, followed by cleavage form the resin by TFA solution andpurification using HPLC to the degree of 95% as described above.

The P33 monomer and dimer, as well as other P33 derivative peptides werecompared for their ability to induce calcium flux in the Mas-transfectedcells at 0.1, 1, 10, 100, 1000 and 10000 nM. As shown in FIG. 9, themonomeric forms of P33 (P33_mono and P33_V) are equally potent while theP33_dimer is superior to the monomeric peptides. P33_(—)5 showed noactivity in this assay (FIG. 9), while the other P33 derivatives(P33_(—)8, P33_(—)9 and P33_(—)10) induced calcium flux, albeit at asomewhat lower degree compared to P33_V (FIG. 10).

B. Activation of Mas by P61 Family

a. Peptide 61

Peptide 61 (SEQ ID NO:9) has a Cysteine in position 5 which wassubstituted for Serine in order to avoid dimerization or otherinteractions of the peptide. The peptide thus obtained was named P61_S(SEQ ID NO:10) (FIG. 1). As shown in FIG. 11, P61_S induced calcium fluxin the Mas-transfected cells: samples of Peptide 61_S present in wells 1and 2 increased calcium flux during the time period between 45 secondsand 90 seconds relative to the negative control whereas the sample ofPeptide 61_S present in well 3 increased calcium flux during the timeperiod between 50 seconds and 90 seconds relative to the negativecontrol.

FIG. 39 shows the dose response of Peptide 61_S. The doses examined inthis experiment were 10, 30, 100, 300, 1000 and 3000 nM. The highestresponse to Peptide 61_S was not reached. The results indicate that theEC50 for Peptide 61_S is at least 500 nM. As explained above, the knownendogenous Mas ligand, Ang (1-7), did not induce a Ca response.

b. P61 Peptide Derivatives

Similarly to P33 (see above), the original cysteine-bearing P61 wassynthesized both as a monomer (P61_mono) and as a dimer (P61_dimer) (SEQID NO. 9). Shorter derivates of P61 were also synthesized (FIG. 1).These P61-related peptides were studied for their ability to inducecalcium flux in the Mas-transfected cells. As shown in FIG. 12, theP61_dimer (SEQ ID NO:9) was more potent than the monomeric P61 peptides:P61_S (SEQ ID NO:10) and P61_mono (SEQ ID NO:9). FIG. 13 indicates thatP61_(—)11S (SEQ ID NO:12) was as potent as P61_S, while P61_(—)4 (SEQ IDNO:11) was somewhat weaker.

Mas activation is not always linked to calcium flux, for example, Masendogenous ligand, Ang 1-7 does not elicite calcium flux. Thus, it isstill possible that the peptides which did not induce calcium flux inthe Mas-transfected cells would activate Mas via other patways. Inaddition, it is possible that the experimental system is notsufficiently sensitive or that these peptides interact with the receptorin a different manner, or via different binding site than the “active”peptides. Thus, this interaction should be further characterized.

Out of the 16 peptides (of families P33 and P61) that were screened bythe calcium flux assay (P61_(—)5S was not tested in this assay due toits low solubility in water), four peptides were chosen to be furtherexamined by ex vivo and in vivo assays (P61_S (SEQ ID NO:10); P61-dimer(SEQ ID NO:9); P33_V (SEQ ID NO:7) and P33-dimer (SEQ ID NO:6)), asdescribed below in Examples 9-12.

EXAMPLE 4 Induction of Calcium Flux in FPRL1-Transfected CHO Cells

The ability of Peptides to change calcium flux was examined in CHO-K1cells co-transfected with FPRL1 and Gα16. Several peptides were found toinduce calcium flux in this experimental system as follows:

Peptide P60_S (SEQ ID NO:8):

As shown in FIG. 14 the sample of P60_S present in all three wellsincreased calcium flux during the time period between about 20 secondsand 90 seconds relative to the negative control.

Peptide P33_V (SEQ ID NO:7):

As shown in FIG. 15, the sample of Peptide 33_V present in all threewells increased calcium flux during the time period between about 20seconds and 90 seconds relative to the negative control.

The P33-derived peptides (see FIG. 1): P33_V (SEQ ID NO:7), P33 (SEQ IDNO:6)-mono, P33 (SEQ ID NO:6)-dimer, P33_(—)5 (SEQ ID NO:27), P33_(—)8(SEQ ID NO:14), P33_(—)9 (SEQ ID NO:15) and P33_(—)10 (SEQ ID NO:16)were tested for their ability to activate FPRL1 using the calcium fluxassay in FPRL1-transfected CHO cells. As shown in FIG. 16, the monomericforms of Peptide 33, P33_V and P33_mono, were similarly potent in theirability to induce calcium flux in the concentrations tested (0.1, 1, 10,100, 1000, 10000 nM) while P33_dimer was inactive. The shorter derivatesof P33 (P33_(—)5, P33_(—)8, P33_(—)9 and P33_(—)10) also failed toinduce calcium flux in the tested concentrations (FIG. 16, and data notshown). The lack of activity of these peptides might be due to reducedpotency; insufficient sensitivity of the experimental system,alternative interaction of the receptor, or activation of otherpathways, which are not related to calcium flux. Thus, this interactionshould be further characterized.

Peptide 94 (SEQ ID NO:5)

As shown in FIG. 17, the sample of P94 (SEQ ID NO:5) present in allthree wells increased calcium flux during the time period between about20 seconds and 90 seconds relative to the negative control.

Shorter peptides derived from Peptide 94 (listed in FIG. 1) weresynthesized and purified as previously described. These peptides failedto induce calcium flux (data not shown). This could result frominsufficient sensitivity of the experimental system, alternativeinteraction of the receptor, or activation of other pathways, which arenot related to calcium flux. Thus, this interaction should be furthercharacterized.

Peptide 58 (SEQ ID NO:1)

As shown in FIG. 18, the samples of P58 (SEQ ID NO:1) present in allthree wells increased calcium flux during the time period between about20 seconds and 90 seconds relative to the negative control.

In order to further characterize P58, shorter peptides derived from itssequence were synthesized and studied in the calcium flux assay. Asshown in FIGS. 19-21, P58 and its derivates P58_(—)4 (SEQ ID NO:2),P58_(—)5 (SEQ ID NO:3) and P58_(—)10 (SEQ ID NO:4), elicited calciumflux in the FPRL1 transfected CHO cells in a dose dependent manner. Allpeptides induced calcium flux via FPRL1, but were less potent than Wpeptide that served as a positive control (FIG. 19-20). W peptide isknown for its very high affinity to FPRL1 (ref?). However, W peptide isan artificial peptide with no beneficial therapeutic activity. The P58peptides were also compared to Ac2-26, a known FPRL1 agonist possessinganti-inflammatory activity, and were found to be more potent ineliciting calcium flux (FIG. 21).

The following P58 derivative peptides did not induce calcium flux in theFPRL1-transfected cells: P58_(—)6 (SEQ ID NO:38), P58_(—)7 (SEQ IDNO:36), P58_(—)12 (SEQ ID NO:37) (FIG. 19-20 and data not shown).However, FPRL1 activation is not always linked to calcium flux; forexample, lipoxin A4 and its analogs evoke anti-inflammatory activity viaFPRL1, but do not eliciting calcium flux. Thus, it is still possiblethat these peptides would activate FPRL1 via other patways. In addition,it is possible that the experimental system is not sufficientlysensitive or that these peptides interact with the receptor in adifferent manner, or via different binding site than thecalcium-inducing peptides.

Out of the 20 peptides screened by the calcium flux assay for FPRL1activation, three peptides were chosen to be further examined in in vivoassays: P58 (SEQ ID NO:1), P58_(—)4 (SEQ ID NO:2) and P58_(—)5 (SEQ IDNO:3).

EXAMPLE 5 Competitive Radioligand Binding Assay of Peptide 58 to FPRL1

The specific binding of Peptide 58 (SEQ ID NO:1) to FPRL1 was analyzedby testing its ability to compete with 0.025 nM [¹²⁵I] WKYMVm (Wpeptide), a known agonist of FPRL1, for binding to FPRL1 in CHOtransfected cells. CKβ8-1 (aa 46-137), another known ligand of FPRL1 wasused as a positive control in this assay (Chiang et al 2006,Pharmacological Reviews 58, 463-487). Peptides were incubated with cellsfor 90 minutes at 25° C., in the presence of Incubation Buffer (50 mMHEPES, pH 7.4, 100 mM NaCl, 5 mM KCl, 5 mM MgCl2, 2 mM Ca Cl2, 0.5% BSA)and the amount of radioactive W peptide was measured. The results inFIG. 22 show inhibition of W peptide binding, and indicate that P58inhibited W peptide binding to FPRL1 in a dose dependent manner, with anIC50 0.189 μM and a Ki 0.0541 μM.

EXAMPLE 6 The In Vivo Effect of Peptide 58 on Zymosan-inducedPolymorphonuclear Leukocyte Influx into Air Pouches

In order to analyze the ability of P58 to exert in vivo effects via theFPRL1 receptor, an acute experimental model of inflammation, theZymosan-induced murine dorsal air pouch model, was used. TheZymosan-induced infiltration of PMNs in this model has previously beenshown to be inhibited by agonists of the FPRL1 receptor, such aslipoxins and Annexin 1-derived peptides (Perretti et al 2002, NatureMedicine 8, 1296-1302).

Animals:

Male out-bred Swiss albino mice were purchased from Harlan, UK (T.O.strain) and maintained on a standard chow pellet diet with tap water adlibitum and a 12:00 h light /dark cycle. All animals were housed for 7days prior to experimentation to allow body weight to reach ˜30 g on theday of the experiment.

Drug Treatment and Experimental Design:

Drugs were stored at −20° C. and defrosted on the day of the experiment.Peptide P58 was provided lyophilized; it was taken back to roomtemperature and dissolved with sterile PBS before use to make an initial1 mg/ml solution. Peptide Ac2-26 (positive control) was also dissolvedwith sterile PBS before use to make an initial 1 mg/ml solution. Vehicleconsisted of sterile pyrogen free PBS (Gibco, cat no. 14190-094). Oncedissolved, P58 (SEQ ID NO:1) and Ac2-26 gave a clear solution. Drugs orvehicle were administered i.v. at a final volume of 200-μl, this volumecontaining the doses described below.

Experimental Schedule:

-   Day −6: injection of 2.5 ml of sterile air for air pouch formation.-   Day −3: injection of 2.5 ml of sterile air for air pouch    maintenance.-   Day 0:-   Time 0—Intravenous administration of vehicle (Group A), P58 (Group B    & C) or Ac2-26 (Group D), immediately before intra-pouch injection    of 1 mg zymosan A (Sigma). Another group of mice received P58 (Group    E), and a control group received vehicle (Group F), directly into    the air-pouch in the absence of zymosan A.-   Time +4 h—Air pouches were washed with 2 ml of ice cold PBS    containing 3 mM EDTA.

Lavage fluids were kept all the time on ice, then used to determine thenumber of migrated leukocytes, by taking an aliquot (100 μl) anddiluting it 1:10 in Turk's solution (0.01% crystal violet in 3% aceticacid). The samples were then vortexed and 10 μl of the stained cellsolution were placed in a Neubauer haematocymometer. Differential cellcount was done using a light microscope (Olympus B061). In view of theirchromatic characteristics and their nucleus and cytoplasm appearance,polymorphonuclear leukocytes (PMN; >95% neutrophils) could be easilyidentified.

Experimental groups:

-   group A, vehicle (200 μl i.v.)+zymosan A (n=8)-   group B, peptide P58 (50-μg i.v.)+zymosan A (n=8)-   group C, peptide P58 (200-μg i.v.)+zymosan A (n=8)-   group D, peptide Ac2-26 (200-μg i.v.)+zymosan A (n=8)-   group E, peptide P58 (100 μg in situ) (n=8)-   group F, vehicle (100 μl in situ) (n=5)    FACS Staining and Analysis:

For FACS analyses, an aliquot of lavage fluid was stained with thePE-conjugated anti-GR-1 monoclonal antibody (1:100 dilution, BDBiosciences; Cat 553128) to mark polymorphonuclear leukocytes. Stainingwas performed at 4° C.

Flow cytometry was performed using FACScan analyser (Becton Dickinson,Cowley, UK) with air-cooled 100 mW argon laser tuned to 488 nm connectedto an Apple Macintosh G3 computer running Cell Quest II software.Forward and sidescatter characteristics were initially used todistinguish between the three distinct cell populations (lymphocytes,monocytes and granulocytes). Cells positive for GR-1 were detected inthe FL2 channel (wavelength of 548 nm). Data are expressed as percentageof positive cells (in relation to the specific mAb). Determination ofpositive and negative populations was performed based on the controlstaining with irrelevant IgG isotype (rat IgG2b) labelled with PE. Oncedetermined, quadrants were rigorously maintained for all analyses.

Statistics:

Data are shown for single mice, and also shown as mean±S.E. of (n) miceper group. Statistical differences were determined by ANOVA, plusStudent Newman Keuls test. A P value <0.05 was taken as significant.

Leukocyte Migration:

Intra-pouch challenge with zymosan A triggered a marked leukocyteaccumulation into the air-pouches, (as determined by differential cellcount). FIG. 23 demonstrates the cumulative data on leukocyteaccumulation. This leukocyte accumulation was inhibited significantly bythe treatment with Ac2-26 (FIG. 23). Administration of P58 (SEQ ID NO:1)reduced the leukocyte accumulation triggered by zymosan A, 38% and 26%of inhibition for 50 μg and 200 μg/mouse, respectively (FIG. 23). Theseresults did not reach statistical significance (comparing with thevehicle-treated zymosan A), probably due to the small number of animalsin each group.

PMNs Migration:

Injection of zymosan A into the air pouch produced a marked neutrophilmigration, as determined by FACS analysis with the Gr1 marker.Leukocytes recovered from air pouches were stained for GR-1 as describedabove. FIG. 24 demonstrates the cumulative data on accumulation ofneutrophils (GR-1⁺ cells). Administration to mice of peptide P58 (SEQ IDNO:1) at 50 μg/mouse inhibited 40% of the neutrophil accumulationtriggered by zymosan A (p<0.05; FIG. 24). This degree of inhibition iscomparable to the 52% of inhibition observed in the group treated withAc2-26 (200 μg/mouse). The treatment with P58 at 200 μg/mouse led to alower degree of inhibition (30%) that did not reach statisticalsignificance, perhaps due to the pharmacodynamics of the peptide.Representative FACS histograms are presented in FIG. 25.

Pro-inflammatory Effects of P58:

The possible pro-inflammatory effects of P58 (SEQ ID NO:1) (100 μg givenlocally) were evaluated by its injection directly into the mouseair-pouch, and compared with the injection of the same volume (100 μl)of vehicle (sterile pyrogen free PBS). Peptide P58 failed to triggerleukocyte or neutrophil accumulation into the mouse air pouch (FIGS. 23and 24), suggesting that this peptide does not possess anypro-inflammatory or chemotactic properties. This result also suggeststhat the peptide preparation was LPS-free.

Conclusions:

This study demonstrates that peptide P58 (SEQ ID NO:1), given at thedose of 50 μg/mouse, is effective in reducing PMN accumulation in anexperimental model of cell recruitment, that is in response to localapplication of zymosan A in the mouse air-pouch cavity. Peptide 58displayed a significant inhibition at 50 μg/mouse, comparable to theinhibition triggered by the peptide Ac2-26, despite lower inhibition wasobserved at 200 μg/mouse. It is uncertain if the dose-response profilefor peptide P58 reflects unfavorable PK or if it is within the nature ofactivation of this endogenous receptor (target for P58). Construction offull dose-response curves (e.g. 10-25-50-100 μg/mouse) would allowbetter assessment of the potency of P58, and possibly also closercomparison to the potency of peptide Ac2-26 and other knownanti-inflammatory drugs (e.g. indomethacin, 10 mg/kg).

EXAMPLE 7 The In Vivo Effect of Peptide 58 and its Shorter Derivatives,Peptide 58_(—)4 and Peptide 58_(—)5, on Polymorphonuclear LeukocyteTrafficking

The anti-inflammatory activity of P58 shorter derivatives was tested inthe same model of acute inflammation used in Example 6, theZymosan-induced murine dorsal air pouch model.

Animals:

Male out-bred Swiss albino mice were purchased from Harlan, UK (T.O.strain) and maintained on a standard chow pellet diet with tap water adlibitum and a 12:00 h light /dark cycle. All animals were housed for 7days prior to experimentation to allow body weight to reach ˜25 g on theday of the experiment.

Drug treatment and Experimental Design:

Drugs were stored at −20° C. and defrosted on the day of the experiment.Peptide P58 (SEQ ID NO:1) was provided lyophilized; it was taken back toroom temperature and dissolved with sterile PBS before use to make aninitial 1 mg/ml solution. Peptide P58-4 (SEQ ID NO:2) was providedlyophilized; it was taken back to room temperature and dissolved withsterile PBS before use to make an initial 327 μg/ml solution. PeptideP58-5 (SEQ ID NO:3) was provided lyophilized; it was taken back to roomtemperature and dissolved with sterile PBS before use to make an initial476 μg/ml solution. Vehicle consisted of sterile pyrogen free PBS(Gibco, cat no. 14190-094). Once dissolved, peptides P58 (SEQ ID NO:1),P58-4 (SEQ ID NO:2) and P58-5 (SEQ ID NO:3) gave a clear solution. Drugsor vehicle were administered i.v. at a final volume of 200 μl in thedoses described below.

Experimental Schedule:

-   Day −6: injection of 2.5 ml of sterile air for air pouch formation.-   Day −3: injection of 2.5 ml of sterile air for air pouch    maintenance.-   Day 0:-   Time 0—Intravenous administration of vehicle (Group A), P58 (SEQ ID    NO:1) (Group B & C), P58-4 (SEQ ID NO:2) (Group D & E), P58-5 (SEQ    ID NO:3) (Group F & G) immediately before intra-pouch injection of 1    mg zymosan A (Sigma).-   Time +4 h—Air pouches were washed with 2 ml of ice cold PBS    containing 3 mM EDTA and 25 U/mL of Heparin.

Lavage fluids were kept all the time on ice, then used to determine thenumber of migrated leukocytes, by taking an aliquot (100 μl) anddiluting it 1:10 in Turk's solution (0.01% crystal violet in 3% aceticacid). The samples were then vortexed and 10 μl of the stained cellsolution were placed in a Neubauer haematocymometer. Differential cellcount was done using a light microscope (Olympus B061). In view of theirchromatic characteristics and their nucleus and cytoplasm appearance,polymorphonuclear leukocytes (PMN; >95% neutrophils) could be easilyidentified.

Experimental Groups:

-   group A, vehicle (200 μl i.v.)+zymosan (n=7)-   group B, peptide P58 (200 μg; 80 nmole i.v.)+zymosan (n=7)-   group C, peptide P58 (50 μg; 20 nmole i.v.)+zymosan (n=7)-   group D, peptide P58-4 (65.4 μg; 80 nmole i.v.)+zymosan (n=7)-   group E, peptide P58-4 (16.35 μg; 20 nmole i.v.)+zymosan (n=7)-   group F, peptide P58-5 (95.2 μg; 80 nmole i.v.)+zymosan (n=7)-   group G, peptide P58-5 (24 μg; 20 nmole i.v.)+zymosan (n=7)    FACS Staining and Analysis:

For FACS analyses, an aliquot of lavage fluid was stained with thePE-conjugated anti-GR-1 monoclonal antibody (1:100 dilution,eBiosciences; Cat 11-5931) to mark polymorphonuclear leukocytes.Staining was performed at 4° C. Flow cytometry was performed usingFACScan analyser (Becton Dickinson, Cowley, UK) with air-cooled 100 mWargon laser tuned to 488 nm connected to an Apple Macintosh G3 computerrunning Cell Quest II software. Forward and sidescatter characteristicswere initially used to distinguish between the three distinct cellpopulations (lymphocytes, monocytes and granulocytes). Cells positivefor GR-1 were detected in the FL2 channel (wavelength of 548 nm). Dataare expressed as percentage of positive cells (in relation to thespecific mAb). Determination of positive and negative populations wasperformed based on the control staining with irrelevant IgG isotype (ratIgG2b) labelled with PE. Once determined, quadrants were rigorouslymaintained for all analyses.

Statistics:

Data are shown for single mice, and also shown as mean±S.E. of (n) miceper group. Statistical differences were determined by ANOVA, plusStudent Newman Keuls test. A P value <0.05 was taken as significant.

Leukocyte Migration:

Intra-pouch challenge with zymosan A triggered a marked leukocyteaccumulation into the air-pouches (as determined by differential cellcount). Administration of P58 (SEQ ID NO:1) reduced the leukocyteaccumulation triggered by zymosan A, 47% and 33% of inhibition for 20nmole and 80 nmole/mouse, respectively (FIG. 26). Administration ofpeptide P58-4 (SEQ ID NO:2), reduced the leukocyte accumulationtriggered by zymosan A, 35% and 47% of inhibition for 20 nmole and 80nmole/mouse, respectively (FIG. 26), while administration of peptideP58-5 (SEQ ID NO:3), a 9 amino acid peptide derived from P58 (SEQ IDNO:1) reduced the leukocyte accumulation triggered by zymosan by 29% at80 nmole, but did not inhibit leukocyte migration when administered at20 nmole/mouse (FIG. 26).

PMN Migration:

Injection of zymosan into the air pouch produced a marked neutrophilmigration, as determined by FACS analysis with the Grl marker. FIG. 27demonstrates the cumulative data on accumulation of neutrophils (GR-1⁺cells). Administration of peptide P58 (SEQ ID NO:1) at 20 nmole/mouse or80 nmole/mouse inhibited the zymosan-induced neutrophil accumulation by50% (p<0.05) or 29%, respectively (FIG. 27). Administration of P58-4(SEQ ID NO:2) at 20 nmole/mouse or 80 nmole/mouse inhibited neutrophilmigration by 35% or 49%, respectively (FIG. 27). P58-5 had a weak effectonly at 20 nmole/mouse (FIG. 27).

Conclusions:

This study demonstrates that peptides P58 (SEQ ID NO:1) and P58-4 (SEQID NO:2) are capable of reducing PMN accumulation in an experimentalmodel of cell recruitment in response to local application of zymosan inthe mouse air pouch cavity, with different potencies and efficacies.P58-5 (SEQ ID NO:3) displayed only a weak effect. Peptide P58 (SEQ IDNO:1) (20 nmole/mose) and peptide P58-4 (SEQ ID NO:2) at 80 nmole/mousedisplayed ˜50% inhibition of neutrophils accumulation in the pouch,which is the maximal effect expected in such biological system,indicating that these peptides show biological activity in an animalmodel of acute inflammation.

EXAMPLE 8 The In Vivo Effect of Peptide 58 (SEQ ID NO:1) and its ShorterDerivative, Peptide 58_(—)4 (SEQ ID NO:2) on Myocardial InfarctFollowing Ischaemia-Reperfusion

This Example relates to testing the ability of P58 and its shorterderivative of the present invention to afford protection in murine acutemyocardial infarct following ischaemia-reperfusion. Known agonists ofthe FRPRL1 receptor have been shown to have a protective effect in amurine model of myocardial ischemia-reperfusion (La et al 2001, FASEB J.15, 2247-2256; Gavins et al 2005, FASEB J. 19, 100-102). This model isthus being used to test the ability of P58 and its shorter derivative toafford protection in murine acute myocardial infarct.

Male Albino mice (˜30 g; n=6/group) are subjected toischemia-reperfusion by occlusion of the LADCA (left anterior descendingcoronary artery) for 25 min (ischemia) followed by reopening of LADCA(reperfusion) for 60 min. Peptides are administered i.v. at onset ofreperfusion at different doses, ranging from 5 to 80 nmoles/mouse. Themyocardial tissue damage is assessed by measuring the infarct size(using p-nitro-blue tetrazolium) and area at risk (using Evans bluedye). The results indicate the ability of the peptides to protectagainst experimental myocardial ischemia-reperfusion.

EXAMPLE 9 The Effect of P61_S, P61-dimer (P61_D), P33_V and P33-dimer(P33_D) in Aortic Rings from Wistar Rats and the Participation of NitricOxide (NO) in this Effect

This Example relates to testing the ability of P61_S, P61-dimer (P61_D),P33_V and P33-dimer (P33_D) peptides of the present invention to exertan NO-dependent vasodilating effect on murine aortic rings. Knownagonists of the Mas receptor, i.e. Ang(1-7) and AVE 0991, have beenpreviously shown to have an NO-dependent vasodilating effect on murineaortic rings, which was also dependent on intact endothelium (Lemos etal 2005, J. Cardiovasc. Pharmacol. 46, 274-279; Santos et al 2003,Hypertension 41, 737-743). The ability of Mas-agonistic peptides of thepresent invention to exert such an effect was tested in this model.

The objectives of this study were as follows:

-   -   1. To determine the effect of P61_S (SEQ ID NO:10), P61_D (SEQ        ID NO:9), P33_V (SEQ ID NO:7) and P33_D (SEQ ID NO:6) on        isolated rings of rat aorta.    -   2. To evaluate the role of endothelium in the vascular effects        of P61_S (SEQ ID NO:10), P61_D (SEQ ID NO:9), P33_V (SEQ ID        NO:7) and P33_D (SEQ ID NO:6) on rings of rat aorta.    -   3. To evaluate the participation of NO (Nitric Oxide) in the        vascular effects of P61_S (SEQ ID NO:10), P61_D (SEQ ID NO:9),        P33_V (SEQ ID NO:7) and P33_D (SEQ ID NO:6) on rings of rat        aorta.

For comparison, the effects of Angiotensin 1-7 [Ang-(1-7)] were alsodetermined.

Test System Animal Information Description

Male Wistar rats of the age of 13-14 weeks (body weight: 250 to 300 g)were used. The rats were exposed to light-dark cycle of 12hs (day—06:00to 18:00; night—18:00 to 06:00) controlled by timer. Rats were killed bydecapitation and exsanguination and tissues were rapidly removed.

Rat Aortic Rings Preparation and Mounting

Rings (3-4 mm) from the descending thoracic aorta, free of adipose andconnective tissue, were set up in gassed (95% O2 and 5% CO2)Krebs-Henseleit solution (mmol/L): NaCl 110.8, KCl 5.9, NaHCO3 25.0,MgSO4 1.07, CaCl₂ 2.49, NaH2PO4 2.33 and glucose 11.51, at 37° C., undera tension of 1.0 g, for 1 hour to equilibrate. The presence offunctional endothelium was assessed by the ability of Acetylcholine (1μM) to induce more than 70% relaxation of vessels pre-contracted withphenylephrine (0.3 μM) (Lemos et al., 2005). When necessary, theendothelium was removed by rubbing the intimal surface with a woodenstick. Mechanical activity, recorded isometrically by a force transducer(Panlab, model number TRI 210, Spain), was fed to an amplifier-recorder(Powerlab 4/20, ADInstruments, Inc.) and to a personal computer equippedwith an analogue-to-digital converter board (AD16JR; World PrecisionInstruments, Inc.), using CVMS data acquisition/recording software(World Precision Instruments, Inc.).

Experimental Protocol

The vasorelaxant activity of peptides—P61_S (SEQ ID NO:10), P61 (SEQ IDNO.9)-dimer (P61_D), P33_V (SEQ ID NO:7) and P33 (SEQ ID NO:6)-dimer(P33_D) were measured in vessels (N=5-8) with or without functionalendothelium pre-contracted to the same tension level (approximately 1.5g of tension) induced by submaximal concentrations of phenylephrine(0.01 μM). For comparison, the effect of Ang-(1-7) was also tested (N=4)

P61_S (SEQ ID NO:10), P61 (SEQ ID NO9)-dimer (P61_D), P33_V (SEQ IDNO:7) or P33 (SEQ ID NO:6)-dimer (P33_D) were added in increasingcumulative concentrations (0.0001 to 1 μM) once the response tophenylephrine had stabilized (FIG. 28). In order to verify theparticipation of endothelium-derived products in the relaxant effect ofpeptides, experiments were performed in the presence of 100 μMN^(ω)-Nitro-L-Arginine Methyl Ester -[L-NAME]- a nonselective inhibitorof nitric oxide synthase. In experiments performed in the presence ofL-NAME, vessels were pre-contracted with 0.03 μM of phenylephrine, toachieve the same tension level as the others. L-NAME was added to thebath 20 min prior to the addition of phenylephrine.

Statistical Analysis

Results are presented as mean±SEM. Two-way analysis of variance (ANOVA)with Bonferroni multiple comparison post-test was used to compareconcentration response curves obtained in aortic rings. The vasodilatoreffect of P61_S (SEQ ID NO:10), P61 (SEQ ID NO9)-dimer (P61_D), P33_V(SEQ ID NO:7), P33 (SEQ ID NO:6)-dimer (P33_D) and Ang-(1-7) wereexpressed as percentage decrease in maximal contraction induced byphenylephrine. All statistical analyses were considered significant whenp<0.05.

Results

In endothelium-containing aortic rings pre-contracted withphenylephrine, P61_S (SEQ ID NO:10) produced a concentration-dependentvasodilator effect (FIG. 29). The vasorelaxation induced by P61_S (SEQID NO:10) was abolished in endothelium-denuded vessels (FIG. 29).Maximal values (%) for the relaxant effect of P61_S (Emax) were39.99±5.034 for vessels with endothelium.

To study the participation of NO in the relaxation induced by P61_S,additional experiments were performed in the presence of the NO synthaseinhibitor, L-NAME. The results, shown in FIG. 30, indicate thatinhibition of NO synthase abolished the vasodilator effect of P61_S.

FIG. 31 shows the endothelium-dependent vasodilator effect induced byP61_D (SEQ ID NO:9) in rat aortic rings. This effect was abolished inthe absence of functional endothelium. Maximal values (%) for therelaxant effect of P61_D (Emax) were 20.45±5.11 for vessel withendothelium.

In order to evaluate whether NO participates in the response induced byP61_D, the vasodilator effect of this peptide was tested in the presenceof L-NAME. As shown in FIG. 32, after blockade of NO synthase, theendothelium-dependent relaxation induced by P61_D (SEQ ID NO:9) wasmarkedly inhibited, although a residual vasorelaxation was observed atthe higher concentrations of P61_D.

FIG. 33 shows the vasodilator effect induced by P33_V (SEQ ID NO:7) inrat aortic rings. This effect was completely dependent on a functionalendothelium. Maximal values (%) for the relaxant effect of P33_V (Emax)were 15.69±3.66 for vessels with endothelium.

To study the participation of NO in the vasorelaxation induced by P33_V(SEQ ID NO:7), its effect was tested in the presence of L-NAME. Afterblockade of NO synthase the endothelium-dependent relaxation induced byP33_V was completely inhibited (FIG. 34).

FIG. 35 shows that P33_D induced vasodilator effect in rat aortic rings.This effect was completely inhibited in the absence of a functionalendothelium. Maximal values (%) for the relaxant effect of P33_D (Emax)were 17.78±3.43 for vessels with endothelium.

To verify the participation of NO in the vasodilator effect of P33_D(SEQ ID NO:6), its effect was tested in the presence of L-NAME. Afterthe inhibition of NO synthase, the effect of P33_D was completelyinhibited (FIG. 36).

FIG. 37 shows the effect induced by Ang-(1-7) in aortic rings fromWistar rats. This effect was abolished in the absence of functionalendothelium. Maximal values (%) for the relaxant effect of Ang-(1-7)(Emax) were 15.64±1.91 and 2.82±3.11 for vessels with and withoutendothelium, respectively.

To study the participation of NO in the relaxation induced by Ang-(1-7),additional experiments were performed in the presence of the NO synthaseinhibitor (L-NAME). After blockade of NO synthase theendothelium-dependent relaxation induced by Ang-(1-7) was completelyinhibited (FIG. 38).

In summary, P61_S, P61_D, P33_V and P33_D induced aconcentration-dependent vasodilator effect in aortic rings from Wistarrats. The response induced by these peptides was dependent on thepresence of endothelium. The effect of the peptides P61_S, P33_V andP33_D was blocked in the presence of L-NAME. The vasodilator effectinduced by P61_D was partially but significantly reduced in the presenceof L-NAME. The results indicate that in the aorta of Wistar rats, thevasodilator effect of P61_S, P61_D, P33_V and P33_D is dependent onendothelium-derived NO.

For comparison, the effect of Ang-(1-7) was also tested. In aorta ofWistar rats Ang-(1-7) induced a vasodilator effect. This response wasdependent on endothelium and NO. These results are in accordance withseveral other reports from the literature showing that the vasodilatoreffect of Ang-(1-7) was dependent on endothelium-derived NO (Le tran &Forster, 1997; Silva et al., 2007).

The peptides P61_S, P61-dimer (P61_D), P33_V, and P33-dimer (P33_D)induced an NO- and endothelium-dependent vasodilator effect in isolatedaortic rings from Wistar rats.

EXAMPLE 10 The Participation of D-Pro⁷-Ang 1-7 Sensitive Mechanism inthe Relaxant Effect of Peptides P61_S, P61_D, P33_V, and P33_D

In order to verify the participation of Mas-specific pathway in the inthe relaxant effect of peptides P61_S (SEQ ID NO:10), P61-dimer (P61_D)(SEQ ID NO9), P33_V (SEQ ID NO:7), P33-dimer (P33_D) (SEQ ID NO:6), theabove-mentioned experiments are repeated in the presence of D-Pro⁷-Ang1-7, a known Mas-specific antagonist (Lemos et al 2005, J. CardiovascPharmacol 46, 274-279; Santos et al. 2003, Hypertension 41, 737-743).

The vasorelaxant activity of peptides is measured in vessels with orwithout functional endothelium pre-contracted to the same tension level(approximately 1.5 g of tension) induced by submaximal concentrations ofphenylephrine (0.03 μM or 0.1 μM). Peptides are added in increasingcumulative concentrations (0.0001 to 1 μM) once the response tophenylephrine had stabilized. In order to verify the participation ofD-Pro⁷-Ang 1-7 sensitive mechanism in the relaxant effect of Peptides,experiments are performed in the presence of D-Pro⁷-Ang 1-7 (10−6 M). Asa control for this protocol, another vessel segment from each rat issimultaneously monitored for Peptides effects alone. As positivecontrol, cumulative concentration response curves to Ang-(1-7) areconstructed in presence and absence of D-Pro⁷-Ang 1-7.

EXAMPLE 11 The Effect of P61_S, P61-dimer (P61_D), P33_V and P33-dimer(P33_D) Following Ischemia-reperfusion of Isolated Rat Hearts

This Example relates to testing the effect of P61_S, P61-dimer (P61_D),P33_V and P33-dimer (P33_D) Following Ischemia-reperfusion of IsolatedRat Hearts. Ang(1-7) has been previously shown to improve thepost-ischemic function of isolated murine hearts, as manifested byparameters such as coronary blood flow, reperfusion arrhytmias andmechanical cardiac function (Ferreira et al 2002, Brazilian J. of Med.And Biol. Res. 35, 1083-1090; Ferreira et al 2001, Hypertension 38,665-668; Mello 2004, J. of Renin-Angiotensin-Aldosterone System 5,203-208).

The effect of the peptides P61_S (SEQ ID NO:10), P61-dimer (P61_D) (SEQID NO9), P33_V (SEQ ID NO:7), P33-dimer (P33_D) (SEQ ID NO:6) on thecoronary blood flow, reperfusion arrhythmias and the mechanical functionof rat isolated perfused hearts is evaluated using the Langendorffpreparation (N=6 for each dose) of adult (12-14 weeks old) Wistar rats.

Animals Acclimatization

The rats are maintained in the animal facilities for at the most 7 daysbefore the beginning of the experiment. The animals are exposed to abrightness circle of light-dark cycle of 12hs (day—06:00 to 18:00;night—18:00 to 06:00) and the room temperature is kept at 23±2° C.

Isolated Heart Technique—Ischemia/Reperfusion

For the isolated perfused heart technique, the rats are decapitated10-15 minutes after intraperitoneal injection of 400 IU heparin, thethorax is opened and the heart is carefully dissected and perfusedthrough an aortic stump with Krebs-Ringer Solution (KRS) containing (inmmol/L): NaCl (118.4), KCl (4.7), KH2PO4 (1.2), MgSO4.7 H2O (1.2),CaCl2.2 H2O (2.5), Glucose (11.7), NaHCO3 (26.5). The perfusion pressureis maintained constant (65 mmHg) at 37±1° C. and constant oxygenation(5% CO2 and 95% O2). A force transducer is attached through a heart clipto the apex of the ventricles to record the contractile force (tension,g) on a computer, through a data-acquisition system (Biopac System,Santa Barbara, Calif.). A diastolic tension of 1 g is applied to thehearts. Electrical activity is recorded by using the data-acquisitionsystem with the aid of two electrodes place directly on the surface ofthe right atrium and left ventricle (bipolar lead). The heart rate iscalculated from the force records. Coronary flow is measured bycollecting the perfusate over a period of 1 minute at regular intervals.After 15 minutes of the equilibration period, the hearts are perfusedwith the peptides solutions for an additional 20 min period. After thisbaseline period the left anterior descending coronary artery (LAD) isligated. The ligature is released after 15 minutes, and reperfusion isperformed for an additional 30 minutes.

Cardiac arrhythmias are defined as the presence of ventriculartachycardia and/or ventricular fibrillation after the ligature of thecoronary artery is released. To obtain a quantitative measurement, thearrhythmias are graded by their duration, with duration of 30 minutesconsidered as irreversible arrhythmia. Therefore, the occurrence ofcardiac arrhythmias for up to 3 minutes is assigned a factor 2; 3 to 6minutes is assigned a factor 4; 6 to 10 minutes is assigned a factor 6;10 to 15 minutes is assigned a factor 8; 15 to 20 minutes is assigned afactor 10; 20 to 25 minutes is assigned a factor 11; and 25 to 30minutes is assigned a factor 12. A value of 2 to 12 is thus obtained ineach experiment and is denoted as arrhythmia severity index (ASI).

-   Experimental Groups: the hearts are perfused with: KRS (control    group, N=6) or KRS containing the peptide (0.04, 1 and 5 nmol/L, N=6    for each dose).-   Statistical Analysis All data are expressed as mean±SEM. The cardiac    function values of each animal are obtained by the average of the    values collected at 5 minutes interval during the baseline and    experimental period. Statistical significance is estimated using    one-way ANOVA followed by Dunnett's post hoc test (GraphPad Prism    4.0). The level of significance is set at P<0.05.

EXAMPLE 12 Effect of P61_S, P61-dimer (P61_D), P33_V and P33-dimer(P33_D) on Cardiac Remodeling Induced by Isoproterenol

The effect of the peptides P61_S (SEQ ID NO:10), P61-dimer (P61_D) (SEQID NO9), P33_V (SEQ ID NO:7), P33-dimer (P33_D) (SEQ ID NO:6) on cardiacremodeling, was tested by determining their effect on heart hypertrophyand fibrosis (deposition of collagen I, III, and fibronectin) induced byIsoproterenol. Losartan (an antagonist of the known angiotensin IIreceptor, AT1, Kucharewicz et al., 2002; Hypertension, (40): 774-779)was used as positive control. The negative control group was treatedwith vehicle only. Additional control groups included rats not-treatedwith isoproterenol which were treated with the peptides, with Losartanor with the vehicle. The analysis included determination of the leftventricle mass and morphometry. In addition, immunofuorescence analysisof fibronectin and collagens I and III deposition was performed byconfocal microcopy.

Animals Acclimatization: Male Wistar rats Age: 13-14 weeks (250 to 300g) were maintained in the animal facilities for 7 days before thebeginning of the experiment. The animals were exposed to a brightnesscircle of light-dark cycle of 12hs (day—06:00 to 18:00; night—18:00 to06:00), and the room temperature was kept at 23±2° C.

Experimental Design and Procedures

Heart failure was induced by daily injections of isoproterenol (2mg/kg/day, subcutaneously) during 7 days. The rats were divided to thefollowing groups: The groups treated with 0.9% NaCl instead ofisoproterenol (i.e. no induction of remodeling) were each N=4, andconsisted of: control (0.9% NaCl subcutaneously plus water by gavage);control-Losartan (0.9% NaCl subcutaneously plus Losartan 1 mg/Kg once aday, by gavage), control-peptide (0.9% NaCl subcutaneously plus P61_S orP61 _D or P33_V or P33_D, 1 ug/hour/Kg by osmotic minipump, for eachpeptide). The groups that were treated with isoproterenol were N=6 each,and consisted of: ISO (isoproterenol plus water by gavage), ISO+Losartan(isoproterenol plus Losartan, 1 mg/kg once a day, by gavage), ISO plusPeptide (Isoproterenol plus peptide P61_S or P61_D or P33_V or P33_D, 1ug/hour/Kg, by osmotic minipump, for each peptide). The final volume ofgavage and subcutaneous injection was approximately 0.5 ml and 0.1 ml,respectively.

Immunostaining and Confocal Microscopy

Immunofluorescence-labeling and quantitative confocal microscopy wasused to investigate the distribution and quantity of collagen types I,III and fibronectin present in the left ventricles. Hearts werecollected from control and Isoproterenol-treated animals, washed inphosphate-buffered saline (PBS) to remove excess blood, and thencryofixed in a −80° C. solution of 80% methanol and 20% dimethylsulfoxide. Samples were stored (i.e. cryosubstituted) at −80° C. for 5-7days, moved to −20° C. for one day, washed three times in absoluteethanol at room temperature, twice in xylene and then embedded inparaffin following standard methods. 5-8 μm thick sections were mountedon slides, deparaffinized with xylene, rehydrated through a gradedseries of ethanol to PBS and then incubated in blocking solution (1% BSAand 0.1% Tween 20 in PBS) at room temperature for 1 hr.

Sections were incubated overnight at 4° C. with one of the followingprimary antibodies: rabbit anti-human collagen type I, rabbit anti-humancollagen type III or rabbit anti-human fibronectin. All antibodies werediluted with 1:10 diluted blocking solution. After 4-5 rinses in PBS,donkey anti-rabbit IgG conjugated with Cy3 (cat #711-165-152, JacksonImmunoResearch Laboratories) or were added for 1 hr in the dark at roomtemperature. Following washes with PBS, sections were mounted in 90%glycerol/10% TRIS 1M, pH 9.0 and viewed with a laser scanning confocalmicroscope (Zeiss 510Meta). Optimal confocal settings (aperture, gainand laser power) were determined at the beginning of each imagingsession and then held constant during the analysis of all the samples.Nuclei were labeled with 4′6-diamidino-2-phenylindole dihydrochloride(DAPI) cat # D1306—Molecular Probes. For quantitative analysis ofcollagens I, III, and fibronectin, the ImageTool 2.0 image analysisprogram was used (see “ddsdx”at the University of Texas Health ScienceCenter at San Antonio website) to measure the fluorescence intensity inimages randomly selected. Images were captured at 12 bit and analyzed inthe gray scale range of 0 to 255. Fluorescence intensity was measured asan average of the area (i.e., the sum of gray values of all pixelsdivided by the number of pixels in the area) and values recorded asarbitrary units (AU). Background fluorescence was measured andsubtracted from the region of interest.

Diameter of Cardiomyocytes Determined by Histological Analysis

The diameter of each cardiomyocyte is also a measure of cardiachypertrophy. In order to measure the diameter of cardiomyocytes uponremodeling by isoproterenol, and the effect of our peptides on thisprocess, left ventricles are left in 4% Bouin fixative for 24 hours atroom temperature. The tissues are dehydrated by sequential washes with70% ethanol, 80% ethanol, 90% ethanol, and 100% ethanol and imbedded inparaffin. Transversal sections (6 μm) are cut starting from the basearea of the left ventricle at intervals of 40 μm and dyed withhematoxilineosin stain. Myocytes diameter are evaluated in tissuesections (3-4 for each animal) using an ocular micrometer calibratedwith a stage micrometer adapted to a light microscope (BX 60, Olympus)at 400× magnification. Only cardiomyocytes cut longitudinally withnuclei and cellular limits visible are used for analysis (an average of30 cardiomyocytes for each slice). The diameter of each myocyte ismeasured across the region corresponding to the nucleus. Fifty to onehundred cardiomyocytes are analyzed for each animal (n=4-6 differentanimals). These results were not obtained yet.

Statistical Analysis

Data was reported as mean±SEM. Statistical analysis for confocalmicroscopy was performed using unpaired Student's t test followed by theMann Whitney test. Unpaired Student's t test is used for the analysis ofcardiomyocytes. p values of 0.05 or less were considered significant.

Results

Hearts from control and Isoproterenol-treated animals were collected,and the left ventricle mass was determined. Results showing the leftventricle to body mass ratio (LV/BW) are presented in in FIGS. 46-50.The Isoproterenol treatment caused an increase in the left ventricle tobody mass ratio in all of the above groups (FIGS. 46-50). None of thepeptides reduced the Isoproterenol induced raise in the left ventricleto body mass ratio. The positive control, Losartan, did not have aneffect either.

The effect on cardiac remodeling can be analyzed more directly bychecking heart hypertrophy as measured by Fibronectin and Collagen I andIII deposition. Thus, this assay, which is more sensitive than theanalysis of left ventricle mass, was carried out. At the time ofsubmission of this application, the data of only 3 out of 6 animals ofeach group was analysed. The partial data obtained for P61_D and P61_Sare shown in FIGS. 40-45 and demonstrates the effect of these twopeptides on cardiac hypertrophy as measured by deposition ofFibronectin, Collagen I and Collagen III following isoproterenol cardiacremodeling. The results indicate that P61_D and P61_S are effective inreducing the heart hypertrophy induced by Isoproterenol, as indicated bydeposition fibronectin; Collagen I and Collagen III (albeit P61_D effectas measured by Fibronectin deposition was not significant, FIG. 40).

The partial data obtained for P33_V was inconsistent (not shown), andthe analysis is to be completed when the data of all animals in thisgroup is obtained. No data were obtained for P33_D at the time ofsubmission of this application.

The descriptions given are intended to exemplify, but not limit, thescope of the invention. Additional embodiments are within the claims.

What is claimed is:
 1. A method of treating an ischemia-reperfusion injury-related disorder in a subject; comprising administering to a subject in need thereof a therapeutically effective amount of: i. a purified peptide twenty-four or fewer amino acids in length, wherein the amino acid sequence of said peptide comprises the amino acid sequence of any one of SEQ ID NOs: 1-4 and 26; ii. or a pharmaceutical composition comprising said peptide and a pharmaceutically acceptable carrier.
 2. The method of claim 1, wherein said peptide is conjugated or fused to a second peptide or polypeptide.
 3. The method of claim 1, wherein said peptide includes a C-terminal amidated amino acid.
 4. The method of claim 1, wherein said peptide is modified at its amino terminus, and wherein said amino terminal modification includes an N-glycated, N-alkylated, N-acetylated or N-acylated amino acid.
 5. The method of claim 1, wherein said peptide is pegylated or sialylated.
 6. The method of claim 1, wherein said peptide is encapsulated.
 7. The method of claim 1, wherein the ischemia-reperfusion injury related disorder is associated with ischemic and post-ischemic events in organs and tissues, and the disorder is selected from the group consisting of thrombotic stroke; myocardial infarction; ischemic renal failure; ischemia-reperfusion injury to the mesenteric microcirculation; ischemic acute renal failure; ischemia-reperfusion injury to the cerebral tissue; post surgical ischemia-reperfusion injury and post-organ transplantation ischemia-reperfusion injury.
 8. The method of claim 1, further comprising treating the subject with an additional therapeutic agent.
 9. The method of claim 8, wherein said peptide or said pharmaceutical composition is administered in combination with said additional therapeutic agent. 